Small-Molecule Hydrophobic Tagging of Fusion Proteins and Induced Degradation of Same

ABSTRACT

The present invention includes compounds that are useful in perturbing or disrupting the function of a transmembrane or intracellular protein, whereby binding of the compounds to the transmembrane or intracellular protein induces proteasomal degradation of the transmembrane or intracellular protein. The present invention further includes a method of inducing proteasomal degradation of a transmembrane or intracellular protein. The present invention further includes a method of identifying or validating a protein of interest as a therapeutic target for treatment of a disease state or condition

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of, and claims priority to, U.S.patent application Ser. No. 13/992,076, filed Jun. 27, 2014, nowallowed, which is a 35 U.S.C. §371 national phase application of, andclaims priority to, International Application No. PCT/US2011/063401,filed Dec. 6, 2011, which claims priority under 35 U.S.C. §119(e) toU.S. Provisional Applications No. 61/420,584, filed Dec. 7, 2010, andNo. 61/530,014, filed Sep. 1, 2011, all of which applications are herebyincorporated by reference in their entireties herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant no.R01AI084140 awarded by National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compounds and compositions which may beused to perturb and/or disrupt the function of a transmembrane orintracellular protein in order to identify or validate that protein as aprotein of interest. In addition to compounds and methods, the presentinvention is also directed to a method of identifying or validating aprotein as a protein of interest for use as a bioactive agent (drug)target for therapy of a disease state or condition.

BACKGROUND OF THE INVENTION

One of the central challenges of chemical biology remains the ability toperturb the function of any intracellular protein using a smallmolecule. While significant strides have been made towards developingindividual ligands to specific proteins, only approximately 300molecular targets for approved drugs have been characterized¹.Furthermore, the fraction of the proteome classified as “undruggable” bycurrent methods is estimated to be about 80%². It is likely that manyappealing drug candidates have yet to be found and that future advancesin drug development will be able to overcome the boundaries of what isthought to be an “undruggable” target^(3,4). Therefore, the challengefor biologists remains to identify those disease-causing drug targets.To this end, advances in deep sequencing, microarray technology andgenome-wide RNAi screens have been employed successfully to identifypromising new drug targets. For instance, genome-wide RNAi screens havebeen employed to identify synthetic lethal interactions with mutatedoncogenes and to identify genes necessary for various pathogenicinfections⁵⁻⁷.

While target identification is an obvious important first step in drugdevelopment, the in vivo validation of these potential targets remains achallenge. This is due in part to the unpredictablepharmacokinetics/pharmacodynamics of any inhibitory compound identifiedbased on in vitro inhibition of protein function. In other words, is thefailure of a small molecule inhibitor to give the desired in vivo resultan unforeseen consequence of its in vivo metabolism or is its targetprotein simply a poor drug target? To address this question, generalmethods are needed to functionally validate whether modulation of aputative disease-relevant protein leads to the desired in vivo result.RNAi offered initial promise for organismal validation of putative drugtargets, however, the delivery and stability of duplex RNA remain majorhurdles in knocking down mRNA expression in a whole animal setting⁸. Inthe absence of a direct ligand for the target protein, there arecurrently three categories of small molecule-based methods to controlthe function of a protein of interest (POI)⁹. First, the plant hormoneauxin can be employed to dimerize a plant E3 ubiquitin ligase (TIR1)with a domain from the AUX/IAA transcriptional repressor (Aid1), whichwhen fused to a POI can be ubiquitinated by proximity to TIR1¹⁰. Thismethod requires fusing the POI to Aid1, along with an introduction ofthe plant E3 ligase TIR1 into cells. A second general method used toderegulate protein function involves dimerization of FKBP12 and theFKBP12-rapamycin binding (FRB) domain from mTOR. It has been shown thata POI can be recruited to the proteasome or to the mitochondrial outermembrane by this method¹¹⁻¹³. Again, at least two fusion proteins mustbe introduced into the cell for this system to function′. Lastly, twodestabilizing domains (DDs), one based on the FKBP12 protein and theother on E. coli DHFR protein^(14,15), have been developed todestabilize a DD-POI fusion protein. The degradation conferring DD canbe stabilized by inclusion of derivatives of FK506¹⁶ (in the case ofmutagenized FKBP12) or the E. coli DHFR inhibitor trimethoprim (in thecase of DHFR), ultimately leading to increased levels of the fusionprotein. While the DD method has been successfully used in severalstudies¹⁷⁻²⁰, it requires the continued presence of the ligand forstable expression of the fusion protein. This requirement can be aconcern when studying developing embryos, which might not receivesufficient stabilizing ligand, or when studying the long term effects ofa POI, in which case the ligand would have to be injected into an animalfor the duration of the study. Also, in the case of the long-termexpression of the POI, one must bear in mind the possible fluctuationsof the POI levels that are due to the intermittent injections of thestabilizing ligand.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is directed to a hydrophobic compound comprising ahydrophobic moiety linked to a reactive linker, preferably a haloalkanereactive linker (i.e., a linker which contains a haloalkane moiety whichis reactive with a halogenase/hydrolase self-labeling tag, such ashalotag) which forms a covalent bond with the fusion protein linking thehydrophobic compound to the fusion protein. Compounds according to thepresent invention are useful for binding a fusion protein wherein thefusion protein comprises a protein of interest (e.g., a potential drugor other physiological target) and a self-labeling tag (such as ahalotag, snaptag, cliptag, ACPtag, MCPtag, among others) which is usefulfor binding the hydrophobic compound to the fusion protein. In preferredaspects of the invention the hydrophobic compound comprises a haloalkanereactive linker to which the hydrophobic moiety may be linked to thefusion protein through action of a halogenase self-labeling tag (e.g.HaloTag) on the haloalkane reactive linker. Once reacted, thehydrophobic moiety is covalently bonded to the fusion protein. It hasbeen unexpectedly discovered that the hydrophobic moiety covalentlylinked to the fusion protein produces degradation of the fusion protein(through interaction/degradation with the protein of interest) resultingin a denaturation of the protein and proteasomal degradation of thefusion protein. The action of the hydrophobic moiety linked to thefusion protein in degrading the fusion protein may be used in assays todetermine the importance of the protein of interest to a biologicalprocess, for example, the modulation of a disease state or conditionsuch as the growth or inhibition of a cancer cell or tissue. Determiningthe importance of the protein of interest may be used to establish theprotein of interest as a potential target of bioactive agents, includingsmall molecule pharmaceutical agents for the treatment of a diseasestate or condition which is modulated by the protein of interest.

In a method according to the present invention to determine if a proteinof interest is a potential bioactive agent (e.g. drug) target, ahydrophobically labeled fusion protein comprising a protein of interestis exposed to cells and the impact of the degradation of thehydrophobically labeled fusion protein in or on the surface of the cellsis measured to determine if the protein of interest is a potential drugtarget (i.e., modulates a disease or condition for which drug or othertherapy may prove useful). In preferred embodiments, this methodcomprises covalently attaching a fusion protein comprising a protein ofinterest and a self-labeling polypeptide to a hydrophobic moiety Thiscan be achieved by expressing the two polypeptides as a fusion protein.

In a first method aspect, the present invention comprises the steps of:

1. Providing a hydrophobically labeled fusion protein wherein saidfusion protein comprises a protein of interest and a hydrophobic moietycovalently linked to said fusion protein wherein said hydrophobic moietyis capable of degrading said fusion protein intracellularly or on thesurface of cells;

2. Exposing cells which utilize said protein of interest to saidhydrophobically labeled fusion protein (e.g. by intracellular expressionof the fusion protein or by exposure of the cells to the fusionprotein), wherein the fusion protein may be optionally and preferablylabeled with the hydrophobic moiety within or on the surface of saidcells by a small molecule that labels self-labeling polypeptide of thefusion protein with a hydrophobic moiety;

3. Measuring the degradation of the fusion protein in or the surface ofthe cells; and

4. Determining if the degradation of the fusion protein modulates thebiological activity of the cells through a change in a phenotypicresponse of the cells (e.g., a change in the growth and/or activity ofthe cells which is identified) consistent with the protein being apotential target for a bioactive agent (e.g. drug) for a disease and/orcondition modulated through said protein of interest.

In preferred aspects, the method according to the present inventionutilizes a fusion protein comprising a protein of interest and aself-labeling polypeptide to hydrophobically label the fusion protein todetermine if the protein of interest is a potential bioactive agent(e.g. drug) target. This method comprises the steps of:

1. Providing a fusion protein comprising a protein of interest and apolypeptide self-labeling tag in vitro or in vivo, includingintracellularly or on the surface of cells;

2. Covalently linking said fusion protein to a compound comprising ahydrophobic group (preferably, other than a fluorescent moiety having aClog P of at least about 1.5) and a reactive linker, wherein thereactive linker is a substrate for the self-labeling tag which whereinsaid hydrophobic group is covalently linked to said fusion protein, thusproducing a hydrophobically labeled fusion protein;

3. Optionally, isolating said hydrophobically labeled fusion protein;

4. Exposing cells which utilize said protein of interest to saidhydrophobically labeled fusion protein;

5. Measuring the degradation of said fusion protein in or on the surfaceof said cells; and optionally

6. Determining if said degradation of said fusion protein modulatesbiological activity of said cells (through a change in a phenotypicresponse of the cells, e.g., a change in the growth and/or activity ofthe cells which is identified) consistent with the protein being apotential target for a bioactive agent (e.g. drug) for a disease and/orcondition modulated through said protein of interest.

In alternative aspects of the invention, the present invention isdirected to a method of inducing degradation of a fusion protein in acell, the method comprising the steps of

1. expressing a fusion protein in a cell wherein said fusion proteincomprises a protein of interest and a self-labeling polypeptide tag;

2. reacting intracellularly or on the surface of said cell saidexpressed fusion protein with a compound comprising a hydrophobic groupand a group reactive with said self-labeling polypeptide tag, whereinsaid compound upon reaction with said self-labeling polypeptide tagforms a covalent bond with said fusion protein to form a hydrophobicallylabeled fusion protein; and

3. allowing said fusion protein to degrade in or on the surface of saidcell.

In preferred aspects of the invention, the fusion protein is producedand hydrophobic labeling of the fusion protein occurs in or on thesurface of the same cells in which the protein of interest is utilizedso that determination of the relevance of the protein of interest occursin the same cells in which fusion protein is produced and the producedfusion protein is hydrophobically labeled. Thus, in certain preferredaspects of the invention the fusion protein is covalently linked to thehydrophobic moiety through the reactive linker in vivo/intracellularlyor on the surface of cells by expressing the fusion proteinintracellularly (including in test animals, such as a mouse, rat orother mammal) and exposing the fusion protein to a compound comprisingthe hydrophobic moiety and reactive linker (e.g., the compound may beadministered in vivo to the test animal or exposed to the cells growingin medium), wherein the hydrophobic moiety linked to the fusion proteinwill cause the fusion protein to degrade intracellularly or on thesurface of cells with a possible resulting and measurable phenyotypicresponse consistent with the the protein of interest being a potentiallyimportant drug target. It is noted that fusion proteins may be producedintracellularly and anchored on the surface of a cell through the use ofsignal and/or anchor peptide sequences which are native to a cell orexpressed with the fusion protein. Such an approach is well known in theart and allows a fusion protein to be expressed and anchored on acellular surface for attachment of a hydrophobic moiety. It iscontemplated that the present invention is applicable to proteins whichfunction on the surface of cells, as well as proteins which functioninternally in cells.

In the present invention, the fusion protein comprises a protein ofinterest and a polypeptide self-labeling tag (e.g. a Halotag, a Snaptag,a Cliptag, a ACP tag or a MCP tag) to which the hydrophobic moiety canbe bound through a reactive linker. In the case of the Halotag fusionprotein, the reactive linker contains a haloalkane group which reactswith the halogenase of the Halotag to produce a covalent bond with thefusion protein. In the case of a Snaptag fusion protein, the reactivelinker contains a benzyl guanine substrate which reacts with the the DNArepair protein O⁶-alkylguanine-DNA alkyltransferase to afford acovalently linked hydrophobic moiety on the fusion protein. In the caseof a Cliptag fusion protein, the reactive linker contains aO2-benzylcytosine moiety in order to afford the covalently linkedhydrophobic moiety on the fusion protein. In the case of a ACP tag, thereactive linker contains a coenzyme A derivative (CoA derivative) whichis covalently bonded through a post-translational modification catalyzedby the acyl carrier protein (ACP) phosphopantetheinyl transferase AcpS(ACP synthase). In the case of a MCP tag (mutant), the reactive linkercontains a coenzyme A derivative which is covalently bonded through apost-translational modification catalyzed by a phosphopantetheinyltransferase Sfp (SFP synthase), but not AcpS. It is noted that the ACPand MCP tags are useful for providing hydrophobically labeled fusionproteins which are unable to penetrate cells—they are limited in theiruse to proteins of interest which are surface proteins.

In the above-described in the measuring step, degraded protein may bequantified by measuring non-degraded or degraded fusion protein in or onthe surface of said cells using standard methods for identifying andquantifying proteins. These methods include, inter alia, using proteinspecific antibodies linked to a reporter, such as a fluorescent or otherreporter, such methods including immunoassay (e.g. ELISA, among others)and immunoblot, absorbance assays, mass spectrometric methods andproteomics methods, among numerous others. Methods for quantifyingspecific proteins in samples are well known in the art and are readilyadapted to methods according to the present invention. Assaying fordegraded protein and the impact of such degradation on the function of acell, for example, the growth and/or proliferation of the cell (e.g.,cell death) or other characteristic (e.g. biological, physiological) ofa cell evidences the importance of the protein of interest to cellulargrowth and function and establishes whether the protein of interest is amodulator of a disease state or condition and thus a potential target(bioactive agent, including drugs) for the treatment of said diseasestate or condition. Identifying a protein of interest as apharmaceutical target will allow the development of assays to identifycompounds and other bioactive agents exhibiting activity as potentialinhibitors and/or agonists of the protein of interest.

In one aspect, compounds according to the present invention may berepresented by the general formula:

Where

is a hydrophobic group other than a reporter group (e.g. afluorescentgroup) having a ClogP of at least about 1.5 or as specifically asotherwise described herein; and

is a linker group having a reactive moiety which reacts with aself-labeling polypeptide tag of a fusion protein comprising saidself-labeling tag and a protein of interest to form a covalent linkbetween said

group and said fusion protein, wherein said hydrophobic group promotesthe degradation of said protein of interest in said fusion proteincovalently linked to said

group.

In alternative embodiments according to the present invention, acompound according to the present invention comprises a compoundaccording to the chemical structure:

Where

is a hydrophobic group other than a fluorescent moiety having a ClogP ofat least about 1.5 or as otherwise specifically described herein;

is a fusion protein comprising a protein of interest and a self-labelingpolypeptide tag linked to said protein of interest in said fusionprotein, said enzyme tag covalently linking said

group to said fusion protein; andL is a chemical linker which covalently binds said

group to said fusion protein, wherein said hydrophobic group promotesthe degradation of said protein of interest comprising a protein ofinterest covalently linked to said

group.

In preferred aspects of the invention,

compounds which may be used to covalently bind a hydrophobic moiety to afusion protein, which preferably contains a self-labeling tag protein,have the chemical structure:

Where

is a hydrophobic group as otherwise described herein;Z is a group which links

to X;

X is a group linking Z to group Y_(R); andY_(R) is a group which is reactive with the fusion protein, preferably aself-labeling tag on said fusion protein, which forms a covalent bondconnecting the hydrophobic group and the fusion protein.

In preferred aspects, Z is absent (a bond), —(CH₂)_(i)—O, —(CH₂)_(i)—S,—(CH₂)_(i)—N—R, a

group wherein X₁Y₁ forms an amide group, or a urethane group, ester orthioester group, or a

groupEach R is H, or a C₁-C₃ alkyl or alkanol group;Each Y is independently a bond, O, S or N—R;and each i is independently 0 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to6, 1, 2, 3, 4 or 5;

In preferred aspects X is a

groupWhere each D is independently a bond (absent),

j is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5;k is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5;preferably k is 1, 2, 3, 4, or 5;m′ is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5;n is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5;X¹ is O, S or N—R, preferably O;Y is the same as above; and

is a bond (absent) or a

group,Where X² is O, S, NR⁴, S(O), S(O)₂, —S(O)₂O, —OS(O)₂, or OS(O)₂O;X³ is O, S, NR⁴; andR⁴ is H or a C₁-C₃ alkyl group, ora pharmaceutically acceptable salt, enantiomer or stereoisomer thereof.

Preferably,

is a

group or an amide group.

In preferred aspects, Y_(R) is a group which is reactive with aself-labeling tag of the fusion protein, wherein the self-labeling tagis preferably a Halotag, a Snaptag, a Cliptag, a ACPtag or a MCPtag.Preferably, the self-labeling tag is a Halotag, and the reactivesubstrate for the Halotag is a haloalkane group which is optionallysubstituted with one or two ether groups, preferably a C₂-C₁₂ chloralkylgroup which is optionally substituted with one (monoether) or two(diether) ether groups, even more preferably, a haloalkyl diether group.In preferred aspects the haloalkyl diether group is according to thechemical structure:

and forms a chemical structure with hydrophobic group and remainingportion of the linker according to the chemical structure:

Where

Z and X are as otherwise described above.

In alternative embodiments, where the fusion protein comprises aself-labeling tag as a Snaptag, Y_(R) is a benzylguanine group

which provides a compound according to the chemical structure:

Where

Z and X are as otherwise described above.

In alternative embodiments where the fusion protein comprises aself-labeling tag as a Cliptag, Y_(R) is a benzylcytosine group

which forms a compound according to the chemical structure:

Where

Z and X are as otherwise described above.

In further alternative embodiments, where the fusion protein comprises aself-labeling tag as a ACPtag or a MCPtag, Y_(R) is a coenzyme Aderivative

which forms a compound according to the chemical structure:

Where

Z and X are as otherwise described above.

Each of the above compounds will produce compounds covalently linked tofusion proteins by action of the self-labeling tag of the fusion proteinon the reactive moiety of the compounds described above.

Representative compounds which are produced by action of a self-labelingtag are represented by the following structure:

Where

Z, X and

are as otherwise described above, and Y_(Rp) is a chemical moiety whichis formed by the action of the fusion protein, preferably theself-labeling tag protein of the fusion protein, on group Y_(R).

In the case of a fusion protein which comprises a halotag self-labelingtag protein, the reaction product is a compound according to thechemical structure:

Where

Z, X and

are as otherwise described above. It is noted that the Y_(Rp) group(represented as the alkyl diether group) forms a covalent bond (througha nitrogen, oxygen or sulfur group represented as a X_(Fp) group) withthe fusion protein.

In the case of a fusion protein which comprises a snaptap or a cliptagself-labeling tag protein, the reaction product is a compound accordingto the chemical structure:

Where

Z, X and

are as otherwise described above. It is noted that the Y_(Rp) group(represented as a benzyl group) forms a covalent bond (through a sulfurgroup as represented) with the fusion protein.

In the case of a fusion protein which comprises a ACP or MCPself-labeling tag protein, the reaction product is a compound accordingto the chemical structure:

Where

Z, X and

are as otherwise described above. It is noted that the Y_(Rp) groupforms a covalent bond (through an oxygen group with the phosphate asrepresented) with the fusion protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a hydrophobic tagging strategy using the HaloTag fusionprotein system. (a) Chemical structures of the representative HaloTagLigands: HyT5, HyT12, HyT13, HyT16, HyT21 and HyT22. (b) HEK 293T cellsexpressing HA-HaloTag-luciferase were treated with indicated compoundsat 1 μM for 24 hours, at which point luciferase assays were performed.

FIG. 2 shows that HyT13 leads to degradation of HaloTag fusion proteins.(a) Flp-In 293 cells expressing HA-EGFP-HaloTag were treated withindicated concentrations of HyT13 for 24 hours. The lysates were probedwith anti-HA and anti-β-actin antibodies. (b) The same cell line as in(a) was treated for the indicated times with 1 μM HyT13. The rightmostsample was treated with HyT13 for 24 hours, after which HyT13-free mediawas provided for 24 hours. (c) The same cell line as in (a) waspretreated with proteasome inhibitors MG132 (10 μM) and YU101 (10 μM)for 1 hour prior to addition of 1 μM HyT13. The lysates were preparedfrom cells 6 hours after HyT13 addition. (d) HeLa cells stablyexpressing EGFP-HaloTag were treated with vehicle or 1 μM HyT13 for 24hours, whereupon the intracellular GFP fluorescence was quantified byflow cytometry. MFI=mean fluorescence intensity. (e) HEK 293T cellsstably expressing indicated transmembrane HA-HaloTag fusion proteinswere treated with 1 μM HyT13 for 24 hours. Shown are representativeimages from at least three experiments; bands were quantified and meandegradation ±SEM is shown. (f) One-cell stage zebrafish embryos wereinjected with 100 ng of HA-HaloTag-Smad5 cRNA, grown to 256-cell stageand then treated with 10 μM HyT13 for 24 hours. Shown are representativeimages from at least three experiments; bands were quantified and meandegradation ±SEM is shown.

FIG. 3 shows the functional validation of HaloTag degradation by HyT13.(a) NIH-3T3 cells were retrovirally infected with a construct expressingeither HA-HaloTag-HRas(G12V) or HA-HaloTag(D106A)-HRas(G12V). The cellswere then treated with vehicle or 1 μM HyT13 for 24 hours. The lysateswere prepared for immunoblotting and the blots were probed with anti-HAand anti-β-actin antibodies. (b) One hundred thousand NIH-3T3 cellsinfected with HA-HaloTag-HRas(G12V) or HA-HaloTag(D106A)-HRas(G12V) wereplated in 10% FBS containing medium onto 10-cm plates. The next day, themedium was replaced with 1% FBS containing medium, along with vehicle or1 μM HyT13. The media was refreshed every 2 days, and the plates werepictured on day 6. Bar, 5 mm. (c) Quantification of foci as described in(b). The number of foci/cm² was counted from three separate plates, witherror bars representing SEM. (d) One hundred thousandHA-HaloTag-HRasG12V-expressing NIH-3T3 cells were injected into theflank of nude mice on day 0. The mice were administered IP injections ofvehicle or HyT13 daily from day 0. Tumor size was measured daily, andthe tumor volume was calculated. Each treatment group employed 7 mice.Error bars represent SEM.

FIG. 4 shows the schematic of HyT13 mediated degradation of HaloTagfusion proteins. A fusion protein composed of a protein of interest andthe HaloTag protein is degraded upon HyT13 treatment by the proteasome.

FIG. 5 shows representative compounds which were synthesized and anumber which were tested.

FIG. 6 shows the concentration curve of HyT13. Flp-In 293 cellsexpressing HA-EGFP-HaloTag were treated with indicated concentrations ofHyT13 for 24 hours. The lysates were probed with anti-HA andanti-β-actin antibodies, with β-actin serving as a loading control.Shown is quantification of three separate experiments, with error barsrepresenting SEM.

FIG. 7 shows the time course of HyT13 activity. Flp-In 293 cellsexpressing HA-EGFP-HaloTag cells were treated for the indicated timeswith 1 μM HyT13 and the lysates were probed with anti-HA andanti-β-actin antibodies. The rightmost sample was treated with HyT13 for24 hours, after which HyT13-free media was provided for 24 hours. Shownis quantification of three separate experiments, with error barsrepresenting SEM.

FIG. 8 shows that compound HyT13 exhibits no toxicity at doses up to 20μM HyT13. HEK293 or HeLa cells were treated with indicatedconcentrations of HyT13 for 24 hours. The oxidation-reduction indicatorResazurin (alamarBlue, Invitrogen) was employed to determine cellviability. The proteasome inhibitor YU101 is toxic to cells at indicatedconcentration and served as a positive control for the assay.

FIG. 9 shows that there was no observed toxicity in mice treated withHyT13. Nude mice were daily IP injected with indicated concentrations ofHyT13 and were monitored for weight gain during the 14-day experiment.Shown is the percent weight gained during the 14-day period for eachtreatment group ±SEM. Each treatment group consisted of 7 mice.

FIG. 10 shows a serum HyT13 determination. Webster Swiss mice receivedIP injections of 25 mg/kg of HyT13. The injection volume was 10 μL,consisting of 5 μL of Cremophor EL excipient and 5 μL of HyT13 in DMSO.Blood was collected from the carotid artery 1 and 24 hours after theinjection. The blood was allowed to coagulate for 10 minutes,centrifuged at 10,000 g for 5 minutes and the serum was pipetted into anew tube. Ten microliters of the serum were used for a bio-reporterassay, consisting of the ability to degrade luciferase activity in HEK293T luciferase-HaloTag cells. The serum concentration of HyT13 wasbased on a concentration curve of HyT13 performed alongside thebio-reporter assay. No degradation activity was observed in serum frommice receiving no HyT13. Each treatment group consisted of three mice.Shown is the mean serum HyT13 level ±SEM.

FIG. 11 shows the results of a number of synthesized HyT compounds onthe degradation of a green fluorescent protein halotag fusion protein ata concentration for each compound at 1 μM.

FIGS. 12a-e show representative immunoblot gel images of several fusionproteins degradations as described in the experimental section of thepresent application.

FIG. 13 shows the immunoblot gel images of HA-HaloTag-HRas(G12V) fusionprotein.

FIG. 14 shows a number of representative hydrophobic moieties which arecovalently linked to fusion proteins as otherwise described herein.

FIG. 15 shows certain prototypical approaches to create hydrophobic tags(ClogP>1.5) according to the present invention having a haloalkanereactive linker so as to be able covalenly link with a bacterialhalogenase (halotag) polypeptide. Hydrophobic tags (HyTs) can beprepared via the coupling of the commercial Promega reactive ligands andRCO₂H, RNH₂, ROH, RCH₂X by standard synthetic chemical techniques.

FIG. 16 shows ten representative polypeptide (amino acid) sequences forhalotag, snaptag, cliptag, ACPtag and MCPtag self-labeling polypeptidetags used in the present invention.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there may be employedconventional chemical synthetic methods, as well as molecular biology,microbiology, and recombinant DNA techniques within the skill of theart. Such techniques are well-known and are otherwise explained fully inthe literature. See, e.g., Sambrook et al, 2001, “Molecular Cloning: ALaboratory Manual”; Ausubel, ed., 1994, “Current Protocols in MolecularBiology” Volumes I-III; Celis, ed., 1994, “Cell Biology: A LaboratoryHandbook” Volumes I-III; Coligan, ed., 1994, “Current Protocols inImmunology” Volumes I-III; Gait ed., 1984, “Oligonucleotide Synthesis”;Hames & Higgins eds., 1985, “Nucleic Acid Hybridization”; Hames &Higgins, eds., 1984,“Transcription And Translation”; Freshney, ed.,1986, “Animal Cell Culture”; IRL Press, 1986, “Immobilized Cells AndEnzymes”; Perbal, 1984, “A Practical Guide To Molecular Cloning.”

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise (such as in the case of a groupcontaining a number of carbon atoms), between the upper and lower limitof that range and any other stated or intervening value in that statedrange is encompassed within the invention. The upper and lower limits ofthese smaller ranges may independently be included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It is to be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set outbelow. It is understood that in the event a specific term is not definedhereinbelow, that term shall have a meaning within its typical usewithin context by those of ordinary skill in the art.

The term “compound”, as used herein, unless otherwise indicated, refersto any specific chemical compound disclosed herein. Within its use incontext, the term generally refers to a single compound comprising ahydrophobic moiety and a linker which is capable of reacting and forminga covalent bond with a fusion protein as otherwise described herein. Incertain instances the term may also refer to stereoisomers and/oroptical isomers (including racemic mixtures) or enantiomericallyenriched mixtures of disclosed compounds. In the present invention incertain instances, especially in preferred aspects of the invention, thecompound contains both a hydrophobic moiety and a linker moiety and ischemically linked through a covalent bond to a fusion protein such thatthe hydrophobic moiety can facilitate and/or produce degradation of theprotein of interest which is part of the fusion protein. Compounds whichare disclosed are those which are stable and where a choice ofsubstituents and claim elements is available, the substituent or claimelement is chosen such that stable compounds are formed from thedisclosed elements and substituents.

The term “patient” or “subject” is used throughout the specificationwithin context to describe an animal, generally a mammal and preferablya human, to whom a treatment or procedure, including a prophylactictreatment or procedure is performed. For treatment of those infections,conditions or disease states which are specific for a specific animalsuch as a human patient, the term patient refers to that specificanimal. In most instances, the patient or subject of the presentinvention is a human patient of either or both genders.

The term “effective” is used herein, unless otherwise indicated, todescribe an amount of a compound or composition which, in context, isused to produce or effect an intended result, whether that resultrelates to the binding of a hydrophobic moiety-linker compound onto afusion protein or the use of a chemically modified fusion protein (towhich is covalently bonded the hydrophobic group). The term effectivesubsumes all other effective amount or effective concentration termswhich are otherwise described or used in the present application.

The term “protein of interest” is used to described inter alia,intracellular and extracellular proteins which exhibit function in or atthe surface of a cell and may be considered drug targets for a diseasestate or condition. Proteins of interest include structural proteins,receptors, enzymes, cell surface proteins, proteins pertinent to theintegrated function of a cell, including proteins involved in catalyticactivity, aromatase activity, motor activity, helicase activity,metabolic processes (anabolism and catrabolism), antioxidant activity,proteolysis, biosynthesis, proteins with kinase activity, oxidoreductaseactivity, transferase activity, hydrolase activity, lyase activity,isomerase activity, ligase activity, enzyme regulator activity, signaltransducer activity, structural molecule activity, binding activity(protein, lipid carbohydrate), receptor activity, cell motility,membrane fusion, cell communication, regulation of biological processes,development, cell differentiation, response to stimulus, behavioralproteins, cell adhesion proteins, proteins involved in cell death,proteins involved in transport (including protein transporter activity,nuclear transport, ion transporter activity, channel transporteractivity, carrier activity, permease activity, secretion activity,electron transporter activity, pathogenesis, chaperone regulatoractivity, nucleic acid binding activity, transcription regulatoractivity, extracellular organization and biogenesis activity,translation regulator activity. Proteins of interest can includeproteins from eurkaryotes and prokaryotes including humans as targetsfor drug therapy, other animals, including domesticated animals,microbials for the determination of targets for antibiotics and otherantimicrobials and plants, and even viruses, among numerous others. Theprotein of interest is one of the two proteins which comprise the fusionprotein of the present invention which protein may be found at the aminoterminus or the carboxylic acid terminus of the fusion protein; theother protein being a reporter protein (e.g., a green fluorescentprotein, a red fluorescent protein, among others), more preferably aself-labeling tag (e.g., Halotag, Snaptag, Cliptag, ACPtag or MCPtag) asotherwise described herein.

The term “fusion protein” or “chimeric protein” as used herein,describes a protein created through the joining of two or more geneswhich originally coded for separate, distinct proteins. Translation ofthe fusion gene results in a single polypeptide (having two polypeptidesegments) with functional properties derived from each of the originalproteins. Fusion proteins according to the present invention areprincipally recombinant fusion proteins and are created artificially byrecombinant DNA technology. In the present invention, the fusionproteins comprise a protein of interest and a second protein, which maybe a reporter protein such as a green or red fluorescent protein or aluciferase protein or preferably, the second protein of the fusionprotein is a self-labeling polypeptide tag protein such as a Halotag,Snaptag, Cliptag, ACPtag or MCPtag, as otherwise described herein. It isnoted that the protein of interest may be positioned at the amino end orthe carboxyl end of the fusion protein and the second protein to which ahydrophobic moiety is linked (e.g. reporter or tag polypeptide) may bepositioned accordingly.

Fusion proteins according to the present invention are recombinantfusion proteins, created through engineering of a fusion gene. Thistypically involves removing the stop codon from a cDNA sequence codingfor the first protein, then appending the cDNA sequence of the secondprotein in frame through ligation or overlap extension PCR, among othertechniques. The introduced DNA sequence will then be expressed alongwith the other DNA sequence by a cell as a single protein. The proteincan be engineered to include the full sequence of both originalproteins, or only a portion of either. If the two entities are proteins,spacer peptides may be added which make it more likely that the proteinsfold independently and behave as expected. In the case where the linkersenable protein purification, spacer peptides in protein or peptidefusions are sometimes engineered with cleavage sites for proteases orchemical agents which enable the liberation of the two separateproteins. Fusion proteins according to the present invention comprise aprotein of interest and a second protein to which a hydrophobic tag maybe linked. As described, fusion proteins according to the presentinvention comprise a protein of interest and a second polypeptide whichfunctions to covalently bind a hydrophobic moiety as otherwise describedherein. The second protein may be, for example, a reporter polypeptidesuch as a fluorescent protein or a luciferasse protein, but in preferredaspects of the invention, the second protein is a self-labelingpolypeptide tag.

Fusion proteins according to the present invention may be created byutilizing commercially available expression vectors which can be used toprepare fusion genes which are created by inserting an appropriate DNAsequence into the expression vector which is introduced into anexpression cell, such as yeast or a bacterial cell in order express thefusion protein. The present invention preferably utilizes fusionproteins which express a self-labeling polypeptide tag as otherwisedescribed herein in addition to the protein of interest in order to linkthe hydrophobic moiety to the fusion protein.

The term “self-labeling polypeptide tag” or “self-labeling tag” is usedto describe a polypeptide tag which is used in preferred fusion proteinsaccording to the present invention as a means to covalently link ahydrophobic moiety to a protein of interest through a linker which isreactive with the self-labeling tag. The self-labeling tag comprises anenzyme (often mutated) which can be inserted into a fusion protein andis reactive with a specific moiety in order to covalently bind a linker(which contains the specific moiety on one end and a hydrophobic moietyon the other end) to the self-labeling tag and consequently, ahydrophobic moiety to the fusion protein. Preferred self-labeling tagsinclude, for example, halotag, snaptag, cliptag, ACPtag and MCPtagself-labeling tags. All of these tags are readily available incommercially available expression vectors from Promega Corporation ofMadison, Wis. (halotag) and New England BioLabs, Inc. of Ipswich,Massachussets, which vectors can accommodate the splicing of a gene fora protein of interest into the expression vector in order to produce thefusion protein comprising a protein of interest and a self-labelingpolypeptide tag.

The halotag self-labeling polypeptide tag is based upon the halotagprotein, a 34 kDa mutated bacterial hydrolase (haloalkane dehalogenase)which has been incorporated into expression vectors by Promegacorporation, which are available commercially. For example, the halotag2self-labeling tag (haloalkane dehalogenase) sequence SEQ ID NO: 1 (seeFIG. 15) may be found at GenBank® Acc. #. AAV70825 and the expressionvector at AY773970. The halotag7 polypeptide is SEQ ID NO:2 (FIG. 16).Halotag is reactive with haloalkanes and when expressed in fusionprotein form, creates a covalent bond between the fusion protein and areactive linker group onto which has been further linked a reportermoiety or, in the present application, a hydrophobic moiety (other thana fluorophore). Although a number of haloalkane groups may be used asthe reactive linker in the halotag system in order to create a covalentbond, the preferred reactive linker is a

group. The halogtag is readily available in commercially availableexpression vectors from Promega Corporation of Madison, Wis. (halotag).These vectors can accommodate the splicing of a gene for a protein ofinterest into the expression vector in order to produce the fusionprotein comprising a protein of interest and a self-labeling polypeptidetag, expressed in E. coli as well as other expression vectors.

The snaptag self-labeling polypeptide tag is based upon a 20 kDa mutantof the DNA repair protein O⁶-alkylguanine-DNA alkyltransferase thatreacts specifically and rapidly with benzylguanine (BG) derivatives asotherwise described herein, leading to irreversible covalent labeling ofthe snaptag with a synthetic hydrophobic moiety containing probe thougha sulfur group residing on the snaptag and the benzyl group of thebenzylguanine synthetic probe. The rate of the reaction of snaptag withBG derivatives is to a large extent independent of the nature of thesynthetic probe attached to BG and permits the labeling of snap fusionproteins with a wide variety of synthetic probes. Expression vectors forincorporating snaptag into numerous fusion proteins (e.g. psnap-tag(m),psnap-tag(m)2, psnap-tag(T7) and psnap-tag (T7)-2 Vector) are availablefrom New England Biolabs, Inc., USA. The polypeptide sequences for eachof the snaptag polypeptides (snaptagm, snaptagm2, snaptagT7 andsnaptagT7-2) are found in FIG. 16 as psnap-tag(m) (SEQ ID NO:3),psnap-tag(m)2 (SEQ ID NO:4), psnap-tag(T7) (SEQ ID NO:5) and psnap-tag(T7)-2 (SEQ ID NO:6).

The cliptag self-labeling polypeptide tag is based upon a mutation ofthe snaptag DNA alkyltransferase enzyme, resulting in differentialsubstrate specificity. In the case of cliptag protein, this proteinreact specifically with O2-benzylcytosine (BC) derivatives forming acovalent bond between a synthetic probe which is attached toO2-benzylcystosine and the cliptag through a sulfur group on the cliptagand the benzyl group on the benzylcytosine derivatives. The SNAP- andCLIP-tag fusion proteins can be labeled simultaneously and specificallywith different synthetic probes in living cells. Expression vectors forincorporating sliptag into numerous fusion proteins (e.g. clip-tag(m)vector is available from New England Biolabs, Inc., USA). Thepolypeptide sequence for the cliptag polypeptide (cliptagm) is found inFIG. 16 as pclip-tag(m) (SEQ ID NO:7).

The use of ACP and MCP tags are somewhat different from the labeling ofsnap and clip fusion proteins, as the ACP and MCP tags are based on anenzyme-catalyzed post-translational modification. In this approach, theprotein of interest is fused to an acyl carrier protein (ACP) and thecorresponding fusion protein is specifically labeled with CoAderivatives through a post-translational modification catalyzed by thephosphopantetheinyl transferase AcpS (SCP synthase). The ACPtag is of asmall size of 9 kDa. The MCPtag, which is a mutant of the ACP tag ofsimilar size is labeled by the phosphopantetheinyl transferase Sfp (Sfpsynthase) but not by ACP synthase, thereby permitting the selectivelabeling of ACP and MCP fusion proteins with different probes in onesample. In contrast to substrates of the halotag, snaptag and cliptag,substrates of the ACPtag (ACPtagm and ACPtagm-2) and MCPtag (MCPtagm)are not cell permeable, although this approach may be readily utilizedwhere the protein of interest is a cell surface protein. Expressionvectors for these tags (pACP-tag(m), pACP-tag(m)-2 and pMCP-tag(m)) areavailable from New England Biologics, Inc., Massachussets, USA. Theseexpression vectors may be used to readily accommodate many proteins ofinterest to provide an assortment of fusion proteins to determine thefunctionality and important of a protein of interest in methodsaccording to the present invention. These vectors can accommodate thesplicing of a gene for a protein of interest into the expression vectorin order to produce the fusion protein comprising a protein of interestand a self-labeling polypeptide tag, expressed in E. coli as well asother expression vectors. The polypeptide sequences for each of theACPtag and MCPtag polypeptides is found in FIG. 16 as pACP-tag(m) (SEQID NO:8), pACP-tag(m)-2 (SEQ ID NO:9) and pMCP-tag(m) (SEQ ID NO:10).

The preferred self-labeling tags for use in the present invention,halotag, snaptag, cliptag, ACPtag and MCPtag can be used to selectivelylabel corresponding fusion proteins with synthetic probes containinghydrophobic moieties as described herein in both cell assay and in vitroapplications.

The term “hydrophobic group” or “hydrophobic moiety” is used to describea hydrophobic group which is covalently linked to a fusion proteinaccording to the present invention which destabilizes and degrades aprotein of interest in the fusion protein such that the fusion proteinbecomes degraded in a cell (proteasomal degradation). In the presentinvention, the hydrophobic group has the following physicochemicalcharacteristics, in particular, as represented by having a ClogP valueof at least about 1.5, at least about 1.75, at least about 2.0, at leastabout 2.25, at least about 2.5, at least about 2.75, at least about 3.0,at least about 3.25, at least about 3.5, at least about 3.75, at leastabout 4.0, at least about 4.25, at least about 4.5, at least about 4.75,at least about 5.0, at least about 5.25, at least about 5.5.

ClogP is a value which may be readily calculated using ClogP software,available from Biobyte, Inc., Claremont, Calif., USA and applied to anycomputer which utilizes Windows, linux or an Apple operating system.ClogP software is readily adaptable to a number of chemical programsincluding ChemDraw programs and related chemical structure drawingprograms. The value ClogP assigns to the hydrophobicity of a chemical ormoiety is based upon a determination of log P n-octanol/water(logP_(OW)), which is the log of the partitition coefficient of amolecule or moiety in octanol and water. ClogP accurately estimateslogP_(OW) numbers and provides a readout of a value which may be readilyapplied to the present invention. Newer versions of ChemDraw software,available from CambridgeSoft, Inc., Cambridge Mass., USA. incorporatethe ability to interface with ClogP software and provide ClogPcalculations, which may readily accomplished by simply drawing amolecule and applying the ClogP calculation app from that software tothe hydrophobic molecule or moiety to be utilized. Thus, according tothe present invention, virtually any hydrophobic moiety may be proposedand chemically synthesized and incorporated into a reactive linker withthe expectation that that moiety when incorporated into a fusion proteinas otherwise disclosed herein, will produce degradation of the fusionprotein (containing a protein of interest) consistent with the method ofthe present invention.

In the present invention, virtually any hydrophobic group having acalculated ClogP value of at least about 1.5 (as otherwise disclosedhereinabove) may be used to facilitate the degradation of the protein ofinterest in the fusion protein. Representative hydrophobic groupsinclude optionally substituted hydrocarbyl groups containing at leastthree carbon atoms, such as optionally substituted C₃-C₃₀ alkyl, alkeneor alkyne groups, including linear, branch-chained or cyclic (includingbi-cyclo, adamantly and fused ring groups) hydrocarbon groups, arylgroups, including aryl groups containing a single ring or 2 or morefused rings (e.g., two, three or four fused rings) such as optionallysubstituted phenyl groups, including optionally substituted naphthylgroups (including 1- or 2-naphthyl groups), optionally substitutedanthracenyl, phenanthrenyl, and phenacenyl (chrysene) groups, optionallysubstituted triphenyl methyl (trityl, methoxytrityl) groups, optionallysubstituted hydrophobic heterocyclic, including heteroaryl groups suchas optionally substituted quinolinyl groups, among others.Representative hydrophobic groups are found in the chemical compoundswhich are presented in attached FIGS. 5 and 14 respectively. One ofordinary skill in the art may readily adapt ClogP software, combinedwith a structural chemical program (e.g. ChemDraw) to readily providehydrophobic moieties useful in the present invention. In addition, theperson of ordinary skill may modify numerous moieties with hydrophobicmoieties to increase the hydrophobicity of the moiety to provide a ClogPvalue significantly greater than 1.5. It is noted that in certaininstances, useful hydrophobic moeties may have values of ClogP less than1.5, but those moities contain substantial steric bulk which compensatesfor the low levels of hydrophobicity. The inclusion of a boranenido-decaborane group (B₁₀H₁₄) substituent on an aryl group such as aphenyl, naphthyl, phenanthrenyl, anthracenyl (paranaphthyl), etc.

The term “hydrocarbon” or “hydrocarbyl” refers to any monovalent radicalcontaining carbon and hydrogen, which may be straight, branch-chained orcyclic in nature. Hydrocarbons include linear, branched and cyclichydrocarbons, including alkyl groups, alkylene groups, saturated andunsaturated hydrocarbon groups (e.g., alkene, alkyne), includingaromatic groups both substituted and unsubstituted.

“Alkyl” refers to a fully saturated monovalent radical containing carbonand hydrogen, and which may be cyclic, branched or a straight chaincontaining from 1 to 30 carbon atoms, from 1 to 20 carbon atoms,preferably 1 to 10 carbon atoms. Examples of alkyl groups are methyl,ethyl, n-butyl, n-hexyl, n-heptyl, n-octyl, isopropyl, 2-methylpropyl,cyclopropyl, cyclo-propylmethyl, cyclobutyl, cyclopentyl,cyclopentylethyl, cyclohexylethyl and cyclohexyl. Preferred alkyl groupsare C₁-C₆ or C₃-C₁₀ alkyl groups. “Alkylene” refers to a fully saturatedhydrocarbon which is divalent (may be linear, branched or cyclic) andwhich is optionally substituted. Other terms used to indicatesubstitutent groups in compounds according to the present invention areas conventionally used in the art.

“Aryl” or “aromatic”, in context, refers to a substituted orunsubstituted monovalent aromatic radical having a single ring (e.g.,benzene) or multiple condensed rings (e.g., naphthyl, anthracenyl,phenanthryl, phenacenyl) and can be can be bound to the compoundaccording to the present invention at any position on the ring(s). Otherexamples of aryl groups, in context, may include heterocyclic aromaticring systems “heteroaryl” groups having one or more nitrogen, oxygen, orsulfur atoms in the ring (moncyclic) such as imidazole, furyl, pyrrole,pyri-dyl, furanyl, thiene, thiazole, pyridine, pyrimidine, pyrazine,triazole, oxazole, indole or preferably fused ring systems (bicyclic,tricyclic), among others, which may be substituted or unsubstituted asotherwise described herein. Preferred heteroaryl groups are hydrophobicin nature or can be rendered hydrophobic by including one or morehydrophobic substituents on the heteroaryl group, or creating a fusedsystem where at least one of the rings is a benzene (phenyl) ring.

The term “cyclic” shall refer to an optionally substituted carbocyclicor heterocyclic group, preferably a 5- or 6-membered ring or fused rings(two, three or four rings) preferably containing from 8 to 14 atoms. Aheterocyclic ring or group shall contain at least one monocyclic ringcontaining between 3 and 7 atoms of which up to four of those atoms areother than carbon and are selected from nitrogen, sulfur and oxygen.Carbocyclic and heterocyclic rings according to the present inventionmay be unsaturated or saturated. Preferred cyclic groups are hydrocarbylgroups, preferably unsaturated hydrocarbyl groups which are optionallysubstituted. Other preferred cyclic groups are bicyclo alkyl groups oradamantly groups, each of which may be optionally substituted. Preferredheterocyclic groups are heteroaryl or heteroaromatic.

The term “heterocyclic group” as used throughout the presentspecification refers to an aromatic (“heteroaryl”) or non-aromaticcyclic group forming the cyclic ring(s) and including at least onehetero atom such as nitrogen, sulfur or oxygen among the atoms formingthe cyclic ring. The heterocyclic ring may be saturated (heterocyclic)or unsaturated (heteroaryl). Exemplary heterocyclic groups include, forexample pyrrolidinyl, piperidinyl, morpholinyl, pyrrole, pyridine,pyridone, pyrimidine, imidazole, indole, quinoline, isoquinoline,quinolizine, phthalazine, naphthyridine, quinazoline, cinnoline,acridine, phenacene, thiophene, benzothiophene, furan, pyran,benzofuran, thiazole, benzothiazole, phenothiazine and carbostyryl, morepreferably pyrrolidinyl, piperidinyl, morpholinyl, pyrrole, pyridine,thiophene, benzothiophene, thiazole, benzothiazole, quinoline,quinazoline, cinnoline and carbostyryl, and even more preferablythiazole, quinoline, quinazoline, cinnoline, carbostyryl, piperazinyl,N-methylpiperazinyl, tetrahydropyranyl, 1,4-dioxane and phthalimide,among others.

Exemplary heteroaryl moieties which may be used in the present invention include for example, pyrrole, pyridine, pyridone, pyridazine,pyrimidine, pyrazine, pyrazole, imidazole, triazole, tetrazole, indole,isoindole, indolizine, purine, indazole, quinoline, isoquinoline,quinolizine, phthalazine, naphthyridine, quinoxaline, quinazoline,cinnoline, pteridine, imidazopyridine, imidazotriazine,pyrazinopyridazine, acridine, phenanthridine, carbazole, carbazoline,perimidine, phenanthroline, phenacene, oxadiazole, benzimidazole,pyrrolopyridine, pyrrolopyrimidine and pyridopyrimidine;sulfur-containing aromatic heterocycles such as thiophene andbenzothiophene; oxygen-containing aromatic heterocycles such as furan,pyran, cyclopentapyran, benzofuran and isobenzofuran; and especiallyaromatic heterocycles comprising 2 or more hetero atoms selected fromamong nitrogen, sulfur and oxygen, such as thiazole, thiadizole,isothiazole, benzoxazole, benzothiazole, benzothiadiazole,phenothiazine, isoxazole, furazan, phenoxazine, pyrazoloxazole,imidazothiazole, thienofuran, furopyrrole, pyridoxazine, furopyridine,furopyrimidine, thienopyrimidine and oxazole. Further heteroaryl groupsmay include pyridine, triazine, pyridone, pyrimidine, imidazole, indole,quinoline, isoquinoline, quinolizine, phthalazine, naphthyridine,quinazoline, cinnoline, acridine, phenacene, thiophene, benzothiophene,furan, pyran, benzofuran, thiazole, benzthiazole, phenothiazine,pyrrolopyrimidine, furopyridine, furopyrimidine and thienopyrimidine,preferably benzothiophene, benzothiazole, quinoline, quinazoline,cinnoline, pyrrolopyrimidine, furopyridine and thienopyrimidine.

The term “substituted” shall mean substituted at a carbon (or nitrogen)position within context, hydroxyl, carboxyl, cyano (C≡N), nitro (NO₂),halogen (preferably, 1, 2 or 3 halogens, especially on an alkyl,especially a methyl group such as a trifluoromethyl), thiol, alkyl group(preferably, C₁-C₁₀ more preferably, C₁-C₆), alkoxy group (preferably,C₁-C₁₀ alkyl or aryl, including phenyl and substituted phenyl), ester(preferably, C₁-C₁₀ alkyl or aryl) including alkylene ester (such thatattachment is on the alkylene group, rather than at the ester functionwhich is preferably substituted with a C₁-C₁₀ alkyl or aryl group),thioether (preferably, C₁-C₁₀ alkyl or aryl), thioester (preferably,C₁-C₁₀ alkyl or aryl), (preferably, C₁-C₁₀ alkyl or aryl), halogen (F,Cl, Br, I), nitro or amine (including a five- or six-membered cyclicalkylene amine, further including a C₁-C₁₀ alkyl amine or C₁-C₁₀ dialkylamine), amido, which is preferably substituted with one or two C₁-C₁₀alkyl groups (including a carboxamide which is substituted with one ortwo C₁-C₁₀ alkyl groups), alkanol (preferably, C₁-C₁₀ alkyl or aryl), oralkanoic acid (preferably, C₁-C₁₀ alkyl or aryl). Preferably, the term“substituted” shall mean within its context of use alkyl, alkoxy,halogen, ester, keto, nitro, cyano and amine (especially including mono-or di-C₁-C₁₀ alkyl substituted amines). Any substitutable position in acompound according to the present invention may be substituted in thepresent invention, but preferably no more than 5, more preferably nomore than 3 substituents are present on a single ring or ring system.Preferably, the term “unsubstituted” shall mean substituted with one ormore H atoms. Preferred substituents are those which have hydrophobiccharacteristics as otherwise described herein. It is noted that theincorporation of a hydrophobic substituent onto an otherwise lesshydrophobic or non-hydrophobic moiety may render the enter moietyhydrophobic as described for the present invention. A preferredsubstituent on aryl groups (e.g., phenyl, naphthyl) for use in thepresent invention is the borane nido-decaborane group (B₁₀H₁₄), whichalthough is not a hydrophobic group per se, provides the favorablecharacteristics of a significant steric effect to enhance degradation offusion proteins in the present invention.

The term “linker” is used to describe a chemical group which covalentlylinks the hydrophobic moiety to the fusion protein in preferred aspectsof the present invention. In particular, the linker binds to thehydrophobic moiety at one end and to the fusion protein at the otherend. In its broadest aspects, the linker may link the hydrophobic moietyto the fusion protein using conventional chemistry, by reacting(condensing) a nucleophilic group on the fusion protein (an amine,sulfhydryl or hydroxyl group) with an electrophilic group (carboxylicacid, etc.) on the linker to which the hydrophobic groups is attached,thus providing a compound which links the hydrophobic moiety to thefusion protein via the linker. In certain preferred embodiments, thelinker binds to a self-labeling tag of the fusion protein by the actionof the self-labeling tag on a reactive portion of the linker (“reactivelinker”), depending upon the type of self-labeling tag. The chemistryassociated with the various linkers according to the present inventionwill be a function of the fusion protein to which the hydrophobic moietyis to be linked, especially in the case where the fusion proteincomprises a self-labeling protein tag (halotag, etc. as otherwisedisclosed herein), in which case the chemistry of the linker willreflect the substrate specificity of the self-labeling protein tag.Because the reactive moiety of the linker is specific to theself-labeling tag used in the fusion protein of the present invention,the chemistry of the linker at that (reactive end) end which covalentlybinds to the fusion protein will be a function of the substratespecificity for that self-labeling tag protein. Thus, the reactivemoiety of the linker is specific as a substrate for the self-labelingtag of the fusion protein, wherein the self-labeling tag is preferably aHALOtag, a SNAPtag, a CLIPtag, a ACPtag or a MCPtag, all well-known inthe art.

Preferably, the self-labeling tag is a HALOtag, in particular ahaloalkane group (preferably a C₂-C₁₂ chloralkyl, even more preferably,a haloalkyl diether group, in preferred aspects a group according to thechemical structure:

In the case of where the fusion protein comprises a self-labeling tag asa SNAPtag, Y_(R) is a benzylguanine group which forms a compoundaccording to the chemical structure:

Where

Z and X are as otherwise described above.

In the case where the fusion protein comprises a self-labeling tag as aCLIPtag, Y_(R) is a benzylcytosine group which forms a compoundaccording to the chemical structure:

Where

Z and X are as otherwise described above.

In the case where the fusion protein comprises a self-labeling tag as aACPtag or a MCPtag, Y_(R) is a coenzyme A derivative which forms acompound according to the chemical structure:

Where

Z and X are as otherwise described above.

The term “connector”, symbolized in compounds according to the presentinvention by the symbol [CON], is used to describe a chemical moietywhich is optionally included in compounds according to the presentinvention in linker groups as otherwise described herein. The connectorgroup is the resulting moiety which forms from the facile condensationof two separate chemical fragments which contain reactive groups whichcan provide connector groups as otherwise described to produce linkergroups which covalent link hydrophobic moieties to fusion proteins incompounds according to the present invention. It is noted that aconnector is distinguishable from a linker in that the connector is theresult of a specific chemistry which is used to provide compoundsaccording to the present invention wherein the reaction product of thesegroups results in an identifiable connector group which forms a linkergroup of greater length as otherwise described herein.

Common connector groups which are used in the present invention includethe following chemical groups:

Where X² is O, S, NR⁴, S(O), S(O)₂, —S(O)₂O, —OS(O)₂, or OS(O)₂O;X³ is O, S, NR⁴; andR⁴ is H or a C₁-C₃ alkyl group.

Compounds according to the present invention are readily synthesizedusing methods well known in the art. In the present invention, apreferred approach to providing a reactive linker with a hydrophobicmoiety covalently linked to same follows well established syntheticchemical methods. A hydrophobic moiety may be derivative and aconvenient approach is to provide a hydrophobic moiety which contains acarboxylic acid or other electrophilic functional group to react with anucleophilic (e.g. amine, hydroxyl or sulfhydryl) group on a linkermolecule to provide a hydrophobic moiety-containing linker. Thehydrophobic linker may contain a reactive moiety to covalently bond thelinker to a fusion protein or the hydrophobic linker may be derivatizedto provide a functional group (e.g., a nucleophilic or electrophilicmoiety) which is capable of reacting with the fusion protein. In thecase of the use of a self-labeling polypeptide tag to covalently linkthe hydrophobic moiety to the fusion protein, the hydrophobic containinglinker is derivatized to contain (preferably, at the distil end awayfrom the hydrophobic moiety) a chemical moiety which acted upon by theself-labeling tag (e.g., halo, snap, clip, ACP or MCP) as otherwisedescribed herein. The formation of the function groups which arereactive with the self-labeling tag is well known and readily providedusing chemical synthetic technicals which are well known in the art. Inthe case of the halotag, the formation of a haloalkane, in particularlypreferred aspects of the present invention, a chloroalkyldiether moietyas otherwise described herein is readily accomplished from commerciallyavailable intermediates. Particular synthetic approaches are provided inthe examples section which follows. In the case of the benzyl guanosineand benzyl cytosine linker analog substrates of snaptag and cliptagself-labeling tags, these are readily provided from reactive linkerswhich are end-capped with benzyl guanosine and benzyl cytosinerespectively.

Once the reactive linker comprising a hydrophobic moiety is provided,reaction with the fusion protein commences to covalently link thehydrophobic moiety to the fusion protein. The reactive linker may becovalently linked to the fusion protein outside of the cell via standardchemical reaction, but preferably is linked via the self-labeling tagintracellularly. The reactive linker and fusion protein may be reactedintracellularly, separate and then utilized in an assay to determine thefunction and importance of the protein of interest in the fusion proteinas a potential target, or alternatively, the fusion protein and reactivelinker may be introduced intracellularly within the same cell in whichthe assay for function and importance takes place. The compoundsaccording to the present invention may be utilized in vitro or in vivo,and may be used in cell-based assays and in animals models, given thatthe relatively low toxicity of many of the the compounds is consistentwith in vivo utilization.

The compounds according to the present invention may be used in cellbased assays to determine the function and importance of a protein ofinterest, by assaying cell function as a consequence of the degradationof the fusion protein to which the hydrophobic moiety is covalentlybonded. These assays may be based upon prokaryote and/or eukaryote cellsand may be directed to animal and plant proteins, as well as microbialproteins, such as fungal and bacterial proteins, as well as viralproteins. Degradation of the fusion protein containing the protein ofinterest may be indicative of the importance of the protein of interestto an important function which modulates a disease state or condition,for example, the growth of cancer cells, an inflammatory response orother biological response, or the proliferation of bacteria and/orviruses. Degradation of the fusion protein under assay conditions may bereadily monitored using one of the many standard techniques available inthe art, including immunoblot, immunoassay (e.g. ELISA, among others),absorbance assays, mass spectrometric methods and proteomics methods,among others. Virtually any technique for measuring proteins may beadapted for use in the present method provided it is otherwiseconsistent with the integrity of the assay performed using compoundsaccording to the present invention.

Elucidating the in vivo function of protein function for drug targetvalidation is a stumbling block in drug development, which may bereadily addressed using the present invention hydrophobic taggingmethodology. For example, many G Protein-Coupled Receptors (GPCRs) GPCRslack a known ligand or function. One could introduce the HaloTag geneinto the mouse genome such that the knock-in transgene encodes a halotagfusion protein fused to a protein of interest, e.g., an orphan GCPR.Administration of a hydrophobic tagged reactive linker to animalsexpressing the fusion protein would induce the degradation of the fusionprotein (by facilitating the covalent linking of the hydrophobic moietycontaining reactive linker to the fusion protein) and the resultingphenotypic response would mimic the effect of a drug (e.g., as aninhibitor of the protein of interest), thus validating the GPCR (or anyother protein) as a drug target.

Another example of the temporal control advantage offered by the presentinvention is in the area of parasite drug target validation. It isdifficult to determine the functional consequence of inhibiting certainparasite proteins due to their complex life cycles, i.e., a proteinmight be needed at two stages, an early one in an animal vector and alatter one in humans. It would be desirable to retain protein functionduring the early stage but then to be able to eliminate it at the laterstage so as to mimic the effects of a human drug against this parasite.By replacing the gene for a particular parasite protein with aself-labeleling tag (e.g., halotag) fused with a candidate gene(producing a protein of interest) and then inducing the degradation ofthis expressed fusion protein using the hydrophobic tagging methodology,one will be able to validate the candidate parasite protein as a drugtarget.

The present invention will now be further described by way of thefollowing examples, the description of which should be taken to merelyexemplify, but not limit, the present invention.

Examples Overview

To develop a general method to degrade any intracellular protein using asmall molecule, we sought to enlist the cellular protein quality controlmachinery. The burial of internal hydrophobic residues within aprotein's core is a major driving force behind protein folding, and,correspondingly, exposure of such hydrophobic regions is considered ahallmark of an unfolded protein²¹⁻²³. For instance, the endoplasmicreticulum Hsp70-class chaperone BiP specifically binds hydrophobic aminoacids and helps slow-folding proteins to fold^(22,24). Should the cellfail to fold the target protein correctly, the unfolded protein iseliminated by either the ubiquitin-proteasome system or autophagy²⁵. Wesought to mimic the partially denatured state of a protein by appendinga hydrophobic tag on its surface in order to induce its degradation. Totest this hypothesis, we selected the HaloTag dehalogenase systemdeveloped by Promega as the fusion protein component²⁶. This system waschosen because HaloTag fusion proteins are commercially available invarious formats and the haloalkane reactive linker binds to the HaloTagdomain covalently, suggesting a high specificity of the ligand forHaloTag. Here, we demonstrate that hydrophobic tagging affords rapid androbust control of the abundance of numerous proteins, includingtransmembrane receptors, in cultured cells as well as in zebrafish andmouse models.

Chemical Synthesis Materials, Purification, and Analysis.

Reagents used for chemical synthesis were purchased from Sigma-AldrichCo. and were used without further purification. All reactions wereperformed in oven-dried or flame-dried glassware fitted with rubbersepta under a positive pressure of nitrogen. THF was distilled fromsodium/benzophenone. Dichloromethane was distilled from calcium hydride.Analytical thin layer chromatography (TLC) was performed using glassplates precoated with silica gel (0.25 mm). TLC plates were visualizedby exposure to UV light (UV), and then were stained by submersion intoaqueous ceric ammonium molybdate (CAM) or ethanolic ninhydrin solution(Ninhydrin) followed by brief heating on hot plate. Flash columnchromatography was performed using silica gel 60 (230-400 mesh, Merck)with the indicated solvents.

¹H and ¹³C spectra were recorded on Bruker Avance DPX-500 or BrukerAvance DPX-400 NMR spectrometers. ¹H NMR spectra are represented asfollows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet,q=quartet, m=multiplet, br=broad), integration, and coupling constant(J) in Hertz (Hz). ¹H NMR chemical shifts are reported relative to CDCl₃(7.26 ppm) and d₄-MeOD (3.30 ppm). ¹³C NMR was recorded relative to thecentral line of CDCl₃ (77.00 ppm) and d₄-MeOD (49.00 ppm). Highresolution mass spectra were measured at the Keck Biotechnology ResourceLaboratory of Yale University. Low resolution mass spectra were acquiredon a Waters Micromass ZQ mass spectrometer or a Perkin-Elmer API 150 EXLCMS spectrometer.

Synthetic Experimental Procedures and Characterization Data Compounds(2, 3, 4, 5, 6) and Control Compound (1)

tert-Butyl (2-(2-hydroxyethoxy)ethyl)carbamate (9)

To a solution of 2-(2-aminoethoxy)-ethanol (2.1 g, 20 mmol) in C₂H₅OH(50 mL) at 0° C. was added Boc₂O (4.36 g, 20 mmol). The reaction mixturewas stirred at rt for 5 h, evaporated, and diluted with CH₂Cl₂ (20 mL)and H₂O (20 mL). The mixture was extracted twice with CH₂Cl₂, and thecombined extracts were washed with brine, dried over Na₂SO₄, filtered,and concentrated. The residue was chromatographed on silica gel tofurnish tert-butyl (2-(2-hydroxyethoxy)ethyl)carbamate 9 (4.09 g,quant.). ¹H NMR (400 MHz, CDCl₃) δ 5.01 (brs, 1H), 3.76-3.72 (m, 2H),3.58-3.54 (m, 4H), 3.35-3.32 (m, 2H), 2.39 (t, J=5.9 Hz, 1H), 1.44 (s,9H). ¹³C NMR (100 MHz, CDCl₃) δ 156.1, 79.3, 72.1, 70.3, 61.7, 40.3,28.7. LRMS (ES+) [M+Na]⁺228.4. TLC (33% EtOAc in hexanes), R_(f) 0.08(Ninhydrin).

tert-Butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate (11)

To a solution of tert-butyl (2-(2-hydroxyethoxy)ethyl)carbamate 9 (2.15g, 10.48 mmol) in THF (20 mL) and DMF (10 mL) at 0° C. added portionwiseNaH (60% dispersion in mineral oil, 560 mg, 14.04 mmol). After stirringat 0° C. for 0.5 h, 6-chloro-1-iodohexane 10 (Sigma-Aldrich, 2.4 mL,15.72 mmol) was added to the mixture at 0° C. The reaction mixture wasstirred at 0° C. for 20 min, at rt for 14 h, and quenched at 0° C. withsaturated NH₄Cl solution in H₂O. The mixture was extracted twice withethyl acetate and the combined extracts were washed with brine, driedover Na₂SO₄, filtered, and concentrated. The residue was chromatographedon silica gel to afford tert-butyl(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl) carbamate 11 (2.4 g, 71%). ¹HNMR (400 MHz, CDCl₃) δ 4.98 (brs, 1H), 3.61-3.51 (m, 8H), 3.46 (t, J=6.7Hz, 2H), 3.31 (t, J=4.7 Hz, 2H), 1.81-1.74 (m, 2H), 1.61-1.57 (m, 2H),1.49-1.33 (m, 4H), 1.43 (s, 9H). ¹³C NMR (125 MHz, CDCl₃) δ 155.9, 79.2,71.2, 70.3, 70.2, 70.0, 45.0, 32.5, 29.4, 28.4, 26.7, 25.4. LRMS (ES+)[M+Na]⁺ 346.3. TLC (33% EtOAc in hexanes), R_(f) 0.36 (Ninhydrin).

2-(2-((6-Chlorohexyl)oxy)ethoxy)ethanamine (8)

To a solution of tert-butyl (2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate 11 (1.348 g, 4.171 mmol) in CH₂Cl₂ (30 mL) at0° C. were added TFA (5 mL). After stirring at 0° C. for 2.5 h, TFA andsolvent were removed in vacuo and the residue was diluted with MeOH (30mL). The solution was cooled to 5° C. and K₂CO₃ (1.65 g, 11.929 mmol)was added to the mixture. The mixture was stirred at the sametemperature for 10 min, filtered, and evaporated. The residue wasdiluted with H₂O (20 mL) and the mixture was extracted four times withethyl acetate. The combined extracts were dried over Na₂SO₄, filtered,and concentrated. The crude amine was purified by flash columnchromatography on silica gel to give2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine 8 (867 mg, 93%). ¹H NMR (400MHz, CDCl₃) δ 6.47 (brs, 1H), 3.69 (t, J=4.9 Hz, 2H), 3.63-3.60 (m, 2H),3.56-3.53 (m, 2H), 3.52 (t, J=6.6 Hz, 2H), 3.44 (t, J=6.8 Hz, 2H), 3.12(t, J=4.9 Hz, 2H), 1.79-1.71 (m, 2H), 1.60-1.53 (m, 2H), 1.46-1.39 (m,2H), 1.36-1.28 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 71.1, 70.1, 69.7,45.0, 39.4, 32.4, 29.1, 26.5, 25.1. LRMS (ES+) [M+H]⁺ 223.8, [M+Na]⁺246.1. TLC (10% CH₃OH in EtOAc), R_(f) 0.08 (CAM).

2-((3r,5r,7r)-Adamantan-1-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)acetamide(HyT13, 3)

To a solution of 1-adamantaneacetic acid 12 (Sigma-Aldrich, 19.5 mg,0.10 mmol, 1.0 equiv.) and 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine 8(23 mg, 0.10 mmol, 1.0 equiv.) in CH₂Cl₂ (1.5 mL) at rt were added HOBt(16 mg, 0.12 mmol, 1.2 equiv.) and DIEA (52 μL, 3.0 equiv.). Thereaction mixture was cooled to 0° C. and EDCI (23 mg, 0.12 mmol, 1.2equiv.) was added to the mixture. The resulting mixture was stirred atrt for 20 h and quenched at 0° C. with H₂O (5 mL). The mixture wasextracted twice with ethyl acetate and the combined extracts were washedwith brine, dried over Na₂SO₄, filtered, and concentrated. The residuewas chromatographed on silica gel to afford 3 (HyT13, 38 mg, 96%). ¹HNMR (400 MHz, CDCl₃) δ 5.89 (brs, 1H), 3.61-3.59 (m, 2H), 3.57-3.50 (m6H), 3.47-3.42 (m, 4H), 1.95 (s, 2H), 1.92 (s, 2H), 1.80-1.73 (m, 2H),1.70-1.56 (m, 13H), 1.48-1.41 (m, 2H), 1.40-1.33 (m, 2H). ¹³C NMR (100MHz, CDCl₃) δ 170.9, 71.2, 70.2, 69.9, 51.7, 45.0, 42.5, 38.9, 36.7,32.7, 32.4, 29.4, 28.6, 26.6, 25.3. HRMS (ES+) calculated forC₂₂H₃₈N₈ClNO₃ [M+H]⁺ 400.2613. found 400.2609. TLC (5% CH₃OH in CH₂Cl₂),R_(f) 0.29 (CAM).

N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2,2-diphenylacetamide (HyT12,2)

HyT12 was synthesized by the same methods as HyT13 (3).

¹H NMR (400 MHz, CDCl₃) δ 7.35-7.24 (m, 10H), 6.15 (s, 1H), 4.91 (s,1H), 3.56-3.47 (m, 10H), 3.42 (t, J=6.7 Hz, 2H), 1.80-1.73 (m, 2H),1.62-1.55 (m, 2H), 1.48-1.41 (m, 2H), 1.39-1.31 (m, 2H). ¹³C NMR (100MHz, CDCl₃) δ 171.8, 139.4, 128.8, 128.6, 127.1, 71.2, 70.2, 69.9, 69.6,59.1, 45.0, 39.4, 32.4, 29.4, 26.6, 25.3. LRMS (ES+) [M+H]⁺ 418.4. TLC(5% CH₃OH in CH₂Cl₂), R_(f) 0.33 (UV, CAM).

N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-(9H-fluoren-9-yl)acetamide(HyT16, 4)

HyT16 was synthesized by the same methods as HyT13 (3).

¹H NMR (400 MHz, CDCl₃) δ 7.75 (d, J=7.5 Hz, 2H), 7.50 (d, J=7.4 Hz,2H), 7.38 (d, J=7.4 Hz, 1H), 7.36 (d, J=7.4 Hz, 1H), 7.29 (dd, J=7.4,1.0 Hz, 1H), 7.28 (dd, J=7.4, 1.0 Hz, 1H), 6.0 (brs, 1H), 4.52 (t, J=7.4Hz, 1H), 3.59-3.53 (m, 6H), 3.51-3.49 (m, 2H), 3.47 (t, J=6.7 Hz, 2H),3.36 (t, J=6.7 Hz, 2H), 2.59 (d, J=7.4 Hz, 2H), 1.72-1.65 (m, 2H),1.52-1.45 (m, 2H), 1.39-1.32 (m, 2H), 1.30-1.24 (m, 2H). ¹³C NMR (100MHz, CDCl₃) δ 171.2, 146.4, 140.6, 127.3, 127.0, 124.5, 119.8, 71.1,70.2, 69.8, 69.6, 45.0, 43.9, 40.9, 39.3, 32.4, 29.3, 26.5, 25.3. LRMS(ES+) [M+H]⁺ 430.5. TLC (5% CH₃OH in CH₂Cl₂), R_(f) 0.36 (CAM).

N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2,2-dicyclohexylacetamide(HyT21, 5)

HyT21 was synthesized by the same methods as HyT13 (3).

¹H NMR (400 MHz, CDCl₃) δ 5.85 (s, 1H), 3.60-3.51 (m, 8H), 3.47-3.51 (m,4H), 1.80-1.72 (m, 2H), 1.71-1.57 (m, 13H), 1.49-1.32 (m, 4H), 1.28-1.03(m, 8H), 0.97-0.88 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 174.2, 71.2,70.2, 70.1, 70.0, 59.4, 45.0, 38.7, 36.4, 32.5, 31.5, 29.6, 29.5, 26.7,26.6, 26.5, 25.4. LRMS (ES+) [M+H]⁺ 430.6. TLC (5% CH₃OH in CH₂Cl₂),R_(f) 0.34 (CAM).

(S)—N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-(4-isobutylphenyl)propanamide(HyT22, 6)

HyT22 was synthesized by the same methods as HyT13 (3).

¹H NMR (500 MHz, CDCl₃) δ 7.18 (d, J=8.0 Hz, 2H), 7.09 (d, J=8.0 Hz,2H), 5.88 (s, 1H), 3.53-3.45 (m, 8H), 3.44-3.36 (m, 5H), 2.44 (d, J=7.2Hz, 2H), 1.87-1.79 (m, 1H), 1.61-1.55 (m, 2H), 1.49 (d, J=7.2 Hz, 3H),1.47-1.41 (m, 2H), 1.38-1.32 (m, 2H), 0.89 (d, J=6.6 Hz, 6H). ¹³C NMR(125 MHz, CDCl₃) δ 174.4, 140.5, 138.5, 129.4, 127.2, 71.2, 70.2, 69.9,69.7, 46.7, 44.9, 39.2, 32.4, 30.1, 29.4, 26.6, 25.3, 22.3, 18.5. LRMS(ES+) [M+H]⁺ 412.6. TLC (10% CH₃OH in CH₂Cl₂), R_(f) 0.54 (UV, CAM).

tert-Butyl(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)carbamate (14)

To a solution of Boc-11-amino-3,6,9-trioxaundecanoic acid 13 (PeptidesInternational Inc., Boc-mini-PEG-3, 200 mg, 0.650 mmol) and2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine 8 (145 mg, 0.650 mmol) inCH₂Cl₂ (4.5 mL) at rt were added HOBt (105 mg, 0.780 mmol) and DIEA (280μL, 1.625 mmol). The mixture was cooled to 0° C. and EDCI (150 mg, 0.780mmol) was added to the mixture. The resulting mixture was allowed to rt,stirred at rt for 20 h, and quenched at 0° C. with H₂O (10 mL). Themixture was extracted twice with ethyl acetate and the combined extractswere washed with brine, dried over Na₂SO₄, filtered, and concentrated.The residue was chromatographed on silica gel to afford tert-butyl(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)carbamate 14(296 mg, 89%). ¹H NMR (400 MHz, CDCl₃) δ 7.18 (brs, 1H), 5.13 (brs, 1H),4.00 (s, 2H), 3.69-3.47 (m, 20H), 3.43 (t, J=6.7 Hz, 2H), 3.31-3.28 (m,2H), 1.79-1.72 (m, 2H), 1.61-1.54 (m, 2H), 1.47-1.40 (m, 2H), 1.42 (s,9H), 1.38-1.32 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 169.9, 155.9, 79.1,71.2, 70.8, 70.5, 70.4, 70.2, 70.1, 69.9, 69.7, 45.0, 40.2, 38.5, 32.4,29.4, 28.3, 26.6, 25.3. LRMS (ES+) [M+H]⁺ 535.5. TLC (10% CH₃OH inCH₂Cl₂), R_(f) 0.48 (CAM).

N-(24-Chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)pent-4-ynamide(15)

To a stirred solution of tert-butyl(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl) carbamate 14(170 mg, 0.332 mmol) in CH₂Cl₂ (2.5 mL) at 0° C. was added TFA (0.5 mL).The reaction mixture was stirred at 0° C. for 2.5 h and concentrated.The crude amine was used for the next reaction without furtherpurification.

To a solution of crude amine (0.330 mmol) and 4-pentynoic acid (32 mg,0.330 mmol) in CH₂Cl₂ (2.5 mL) at rt were added HOBt (54 mg, 0.396 mmol)and DIEA (150 μL, 0.825 mmol). The mixture was cooled to 0° C. and EDCI(76 mg, 0.396 mmol) was added to the mixture. The resulting mixture wasallowed to rt, stirred at rt for 17 h, and quenched at 0° C. with H₂O (5mL). The mixture was extracted three times with ethyl acetate and thecombined extracts were washed with brine, dried over Na₂SO₄, filtered,and concentrated. The residue was chromatographed on silica gel toprovideN-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)pent-4-ynamide15 (140 mg, 86%). ¹H NMR (400 MHz, CDCl₃) δ 7.18 (brs, 1H), 6.71 (brs,1H), 4.02 (s, 2H), 3.70-3.64 (m, 4H), 3.63-3.59 (m, 6H), 3.57-3.54 (m,6H), 3.53-3.48 (m, 4H), 3.47-3.42 (m, 4H), 2.54-2.49 (m, 2H), 2.43-2.39(m, 2H), 1.99 (t, J=2.6 Hz, 1H), 1.80-1.72 (m, 2H), 1.62-1.55 (m, 2H),1.48-1.40 (m, 2H), 1.39-1.31 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 171.2,170.3, 83.1, 71.2, 70.6, 70.5, 70.3, 70.2, 70.1, 70.0, 69.9, 69.7, 69.1,45.0, 39.3, 38.6, 35.0, 32.4, 29.4, 26.6, 25.3, 14.8. LRMS (ES+) [M+H]⁺515.62. TLC (5% CH₃OH in CH₂Cl₂), R_(f) 0.42 (UV, CAM).

tert-Butyl (2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamate (16)

To a solution of 11-azido-3,6,9-trioxaundecan-1-amine (Fluka, 370 mg,1.695 mmol) in C₂H₅OH (3.5 mL) at 0° C. was added Boc₂O (370 mg, 1.695mmol). The reaction mixture was stirred at rt for 12 h and evaporated.The residue was diluted with CH₂Cl₂ (5 mL) & H₂O (5 mL) and the mixturewas extracted twice with CH₂Cl₂. The combined extracts were washed withbrine, dried over Na₂SO₄, filtered, and concentrated. The crude residuewas purified by flash chromatography on silica gel to provide tert-butyl(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamate 16 (518 mg, 96%).¹H NMR (400 MHz, CDCl₃) δ 5.01 (s, 1H), 3.69-3.59 (m, 10H), 3.53 (t,J=5.1 Hz, 2H), 3.38 (t, J=5.1 Hz, 2H), 3.32-3.29 (m, 2H), 1.43 (s, 9H).TLC (10% CH₃OH in CH₂Cl₂), R_(f) 0.49 (CAM).

tert-Butyl(2-(2-(2-(2-(4-(28-chloro-3,15-dioxo-7,10,13,19,22-pentaoxa-4,16-diazaoctacosyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)carbamate (17)

To a solution of tert-butyl(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamate 16 (37 mg, 0.116mmol) andN-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)pent-4-ynamide15 (57 mg, 0.116 mmol) in t-BuOH—H₂O (1:1, 0.5 mL) and THF (0.5 mL) atrt were added CuSO₄.5H₂O (3 mg, 0.012 mmol) and sodium ascorbate (1.0 Min H₂O, 3 drops). The reaction mixture was stirred at rt for 22 h andevaporated. The residue was diluted with H₂O (5 mL) and the mixture wasextracted three times with ethyl acetate. The combined extracts werewashed with brine, dried over Na₂SO₄, filtered, and concentrated. Thecrude residue was purified by flash chromatography on silica gel to givetert-butyl(2-(2-(2-(2-(4-(28-chloro-3,15-dioxo-7,10,13,19,22-pentaoxa-4,16-diazaoctacosyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)carbamate 17 (86 mg, 92%). ¹H NMR (400 MHz, CDCl₃) δ7.54 (s, 1H), 7.17 (brs, 1H), 6.60 (brs, 1H), 5.11 (brs, 1H), 4.49 (t,J=5.1 Hz, 2H), 4.02 (s, 2H), 3.84 (t, J=5.1 Hz, 2H), 3.70-3.38 (m, 34H),3.31-3.28 (m, 2H), 3.03 (t, J=7.4 Hz, 2H), 2.61 (t, J=5.1 Hz, 2H),1.79-1.72 (m, 2H), 1.61-1.54 (m, 2H), 1.47-1.40 (m, 2H), 1.42 (s, 9H),1.39-1.31 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 172.0, 170.1, 155.9,146.3, 122.6, 79.1, 77.2, 71.2, 70.65, 70.62, 70.54, 70.51, 70.47,70.42, 70.3, 70.2, 70.16, 70.14, 69.9, 69.8, 69.7, 69.4, 50.1, 45.0,40.2, 39.2, 38.5, 35.5, 32.4, 29.4, 28.4, 26.6, 25.3, 21.4. LRMS (ES+)[M+Na]⁺833.48. TLC (5% CH₃OH in CH₂Cl₂), R_(f) 0.25 (CAM).

3-(1-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)propanamide(HyT5, 1)

To a solution of tert-butyl (2-(2-(2-(2-(4-(28-chloro-3,15-dioxo-7,10,13,19,22-pentaoxa-4,16-diazaoctacosyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)carbamate 17 (30 mg, 0.037 mmol) in CH₂Cl₂ (2.0 mL) at 0° C. were addedTFA (0.5 mL). After stirring at 0° C. for 2.5 h, TFA and solvent wereremoved in vacuo and the residue was diluted with MeOH (0.5 mL). Thesolution was cooled to 5° C. and K₂CO₃ (26 mg, 0.185 mmol) was added tothe mixture. The mixture was stirred at the same temperature for 30 minand extracted three times with ethyl acetate. The combined extracts werewashed with brine, dried over Na₂SO₄, filtered, and concentrated. Thecrude residue was purified by flash chromatography on silica gel to givethe proposed structure of3-(1-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)propanamide 1 (HyT5, 24.5 mg, 93%). ¹H NMR (400MHz, CD₃OD) δ 7.77 (s, 1H), 4.53 (t, J=5.1 Hz, 2H), 3.99 (s, 2H), 3.86(t, J=5.1 Hz, 2H), 3.70-3.50 (m, 28H), 3.46 (t, J=6.5 Hz, 2H), 3.41 (t,J=5.6 Hz, 2H), 3.35 (t, J=5.4 Hz, 2H), 3.12 (t, J=5.0 Hz, 2H), 2.98 (t,J=7.5 Hz, 2H), 2.56 (t, J=7.6 Hz, 2H), 1.79-1.72 (m, 2H), 1.61-1.54 (m,2H), 1.49-1.43 (m, 2H), 1.42-1.35 (m, 2H). ¹³C NMR (100 MHz, CD₃OD) δ174.6, 172.7, 147.6, 124.1, 72.1, 71.8, 71.5, 71.4, 71.37, 71.33, 71.29,71.22, 71.1, 70.6, 70.47, 70.40, 67.9, 51.2, 45.7, 40.6, 40.3, 39.8,36.2, 33.7, 30.5, 27.7, 26.5, 22.5. LRMS (ES+) [M+H]⁺ 711.36, [M+Na]⁺733.36. TLC (10% CH₃OH in CH₂Cl₂), R_(f) 0.09 (Ninhydrin, CAM).

(4-Adamantan-1-yl-phenoxy) acetic acid ethyl ester (18)

To a solution of 4-(1-adamantyl)phenol (250 mg, 1.095 mmol) in DMF (2mL) at rt were added ethyl bromoacetate (150 μL, 1.314 mmol) and K₂CO₃(454 mg, 3.285 mmol). The reaction mixture was stirred at rt for 20 hand diluted with H₂O (10 mL) and ethyl acetate (10 mL). The mixture wasextracted twice with ethyl acetate and the extracts were washed withsat. NaHCO₃ and brine. The combined organic layers were dried overNa₂SO₄, filtered, and concentrated. The crude residue was purified byflash chromatography on silica gel to give (4-adamantan-1-yl-phenoxy)acetic acid ethyl ester 18 (335 mg, quant.) as a white solid. ¹H NMR(500 MHz, CDCl₃) δ 7.27 (d, J=8.7 Hz, 2H), 6.86 (d, J=8.7 Hz, 2H), 4.59(s, 2H), 4.27 (q, J=7.1 Hz, 2H), 2.08 (s, 3H), 1.87 (d, J=2.4 Hz, 6H),1.79-1.71 (m, 6H), 1.30 (t, J=7.0 Hz, 3H). ¹³C NMR (125 MHz, CDCl₃) δ169.1, 155.6, 144.7, 125.9, 114.1, 65.5, 61.2, 43.3, 36.7, 35.6, 28.9,14.1. TLC (10% CH₃OH in CH₂Cl₂), R_(f) 0.48 (UV, CAM).

(4-Adamantan-1-yl-phenoxy) acetic acid (19)

To a solution of ester 18 (290 mg, 0.923 mmol) in THF-H₂O (3 mL/3 mL) atrt was added LiOH.H₂O (454 mg, 3.285 mmol). The reaction mixture wasstirred at rt for 15 h and THF was removed in vacuo. The aqueous mixturewas diluted with H₂O (5 mL), cooled to 0° C., and adjusted to pH 4 with1N—HCl. The mixture was extracted twice with ethyl acetate and theextracts were washed with brine. The combined organin layers were driedover Na₂SO₄, filtered, and concentrated. The crude residue wassolidified with hexanes and the solid was filtered with hexanes anddried in vacuo to furnish (4-adamantan-1-yl-phenoxy) acetic acid 19 (246mg, 93%) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ 7.26 (d, J=8.8 Hz,2H), 6.84 (d, J=8.8 Hz, 2H), 4.60 (s, 2H), 2.05 (s, 3H), 1.89 (s, 6H),1.83-1.75 (m, 6H).

3-(1-(1-(4-((3r,5r,7r)-Adamantan-1-yl)phenoxy)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)-1H-1,2,3-triazol-4-yl)-N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)propanamide(HyT6, 20)

To a solution of (4-adamantan-1-yl-phenoxy) acetic acid 19 (6.3 mg,0.022 mmol) in DMF (0.5 mL) at rt were added HATU (10 mg, 0.027 mmol)and DIEA (10 μL, 0.055 mmol). The mixture was stirred at rt for 0.5 hand a solution of HyT5 1 (16.5 mg, 0.023 mmol) in DMF (0.5 mL) was addedto the mixture. The resulting mixture was stirred at rt for 22 h, andquenched at 0° C. with H₂O (5 mL). The mixture was extracted three timeswith ethyl acetate and the combined extracts were washed with brine,dried over Na₂SO₄, filtered, and concentrated. The residue waschromatographed on silica gel to provide HyT6 20 (19 mg, 88%). ¹H NMR(500 MHz, CD₃OD) δ 7.74 (s, 1H), 7.26 (d, J=8.8 Hz, 2H), 6.87 (d, J=8.8Hz, 2H), 5.46 (s, 1H), 4.46 (t, J=4.9 Hz, 2H), 4.45 (s, 2H), 3.98 (s,2H), 3.80 (t, J=5.1 Hz, 2H), 3.65 (s, 4H), 3.63-3.61 (m, 2H), 3.59-3.51(m, 20H), 3.49 (t, J=5.5 Hz, 2H), 3.45-3.42 (m, 4H), 3.39 (t, J=5.5 Hz,2H), 3.32 (t, J=5.2 Hz, 2H), 2.95 (t, J=7.6 Hz, 2H), 2.52 (t, J=7.6 Hz,2H), 2.04 (s, 3H), 1.86 (d, J=2.3 Hz, 6H), 1.80-1.70 (m, 8H), 1.57-1.52(m, 2H), 1.46-1.40 (m, 2H), 1.38-1.32 (m, 2H). ¹³C NMR (125 MHz, CD₃OD)δ 174.7, 127.0, 124.2, 115.4, 72.2, 71.8, 71.6, 71.5, 71.4, 71.32,71.30, 71.2, 71.1, 70.8, 70.5, 70.4, 51.3, 45.7, 44.5, 40.3, 39.9, 39.8,37.8, 36.7, 36.3, 33.7, 30.5, 30.4, 27.7, 26.5, 22.5. HRMS (ES+)calculated for C₄₉H₈₀N₆O₁₂Cl [M+H]⁺ 979.5523. found 979.5529. TLC (10%CH₃OH in CH₂Cl₂), R_(f) 0.51 (UV, CAM).

To a solution of 3,3,3-triphenyl propionic acid (11.3 mg, 0.0373 mmol)in DMF (0.5 mL) at rt were added HATU (17 mg, 0.0447 mmol) and DIEA (16μL, 0.0932 mmol). The mixture was stirred at rt for 0.5 h and a solutionof HyT5 1 (28 mg, 0.0392 mmol) in DMF (0.5 mL) was added to the mixture.The resulting mixture was stirred at rt for 20 h, and quenched at 0° C.with H₂O (6 mL). The mixture was extracted three times with ethylacetate and the combined extracts were washed with brine, dried overNa₂SO₄, filtered, and concentrated. The residue was chromatographed onsilica gel to affordN-(2-(2-(2-(2-(4-(28-chloro-3,15-dioxo-7,10,13,19,22-pentaoxa-4,16-diazaoctacosyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-3,3,3-triphenylpropanamide21 (HyT7, 33.5 mg, 91%). ¹H NMR (500 MHz, CD₃OD) δ 7.75 (s, 1H), 7.37(brs, 1H), 7.27-7.20 (m, 12H), 7.16-7.13 (m, 3H), 4.47 (t, J=5.0 Hz,2H), 4.00 (s, 2H), 3.82 (t, J=5.0 Hz, 2H), 3.68-3.47 (m, 26H), 3.46-3.40(m, 6H), 3.34 (t, J=5.1 Hz, 2H), 3.15 (t, J=5.2 Hz, 2H), 3.03-3.01 (m,2H), 2.96 (t, J=7.5 Hz, 2H), 2.54 (t, J=7.3 Hz, 2H), 1.77-1.71 (m, 2H),1.58-1.53 (m, 2H), 1.46-1.41 (m, 2H), 1.38-1.34 (m, 2H). ¹³C NMR (125MHz, CD₃OD) δ 174.7, 172.9, 172.7, 148.3, 147.5, 130.5, 128.7, 127.1,124.2, 72.1, 71.7, 71.4, 71.36, 71.33, 71.2, 71.18, 71.13, 70.9, 70.4,70.3, 70.2, 57.5, 51.2, 48.1, 45.7, 40.3, 40.0, 39.8, 36.3, 33.7, 30.5,27.7, 26.4, 22.4. HRMS (ES+) calculated for C₅₂H₇₆N₆O₁₁Cl [M+H]⁺995.5261. found 995.5265. TLC (10% CH₃OH in CH₂Cl₂), R_(f) 0.62 (UV,CAM).

N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-3,3,3-triphenylpropanamide(HyT8, 22)

HyT8 was synthesized by the same methods as HyT13 (3).

¹H NMR (400 MHz, CDCl₃) δ 7.31-7.26 (m, 12H), 7.23-7.19 (m, 3H), 5.36(brs, 1H), 3.58 (s, 2H), 3.53 (t, J=6.7 Hz, 2H), 3.48-3.46 (m, 2H),3.45-3.41 (m, 4H), 3.22-3.20 (m, 2H), 3.16-3.13 (m, 2H), 1.81-1.74 (m,2H), 1.63-1.56 (m, 2H), 1.49-1.42 (m, 2H), 1.40-1.32 (m, 2H). ¹³C NMR(100 MHz, CDCl₃) δ 170.3, 146.3, 129.1, 127.9, 126.2, 71.2, 70.0, 69.8,69.3, 56.1, 48.4, 45.0, 38.9, 32.4, 29.3, 26.6, 25.3. HRMS (ES+)calculated for C₃₁H₃₉NO₃Cl [M+H]⁺ 508.2618. found 508.2617. TLC (10%CH₃OH in CH₂Cl₂), R_(f) 0.69 (UV, CAM).

tert-Butyl(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)amino)-6-oxohexyl)carbamate23

To a solution of 6-(Boc-amino)-caproic acid (28 mg, 0.121 mmol) andamine 8 (27 mg, 0.121 mmol) in CH₂Cl₂ (1.5 mL) at rt were added HOBt (20mg, 0.145 mmol, 1.2 equiv.) and DIEA (63 μL, 0.363 mmol). The reactionmixture was cooled to 0° C. and EDCI (28 mg, 0.145 mmol, 1.2 equiv.) wasadded to the mixture. The resulting mixture was stirred at rt for 20 hand quenched at 0° C. with H₂O (5 mL). The mixture was extracted twicewith ethyl acetate and the combined extracts were washed with brine,dried over Na₂SO₄, filtered, and concentrated. The residue waschromatographed on silica gel to afford tert-Butyl(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl) amino)-6-oxohexyl)carbamate23 (45 mg, 85%). ¹H NMR (400 MHz, CDCl₃) δ 6.09 (s, 1H), 4.59 (s, 1H),3.60-3.49 (m, 8H), 3.46-3.40 (m, 4H), 2.15 (t, J=7.4 Hz, 2H), 1.78-1.71(m, 2H), 1.66-1.55 (m, 4H), 1.50-1.39 (m, 4H), 1.41 (s, 9H), 1.37-1.27(m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 172.8, 155.9, 79.0, 71.2, 70.1,69.9, 69.7, 44.9, 39.0, 36.4, 32.4, 29.7, 29.3, 28.3, 26.6, 26.3, 25.3,25.2. TLC (10% CH₃OH in CH₂Cl₂), R_(f) 0.46 (UV, CAM).

N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-6-(3,3,3-triphenylpropanamido)hexanamide(HyT9, 24)

To a stirred solution of tert-Butyl(6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)amino)-6-oxohexyl) carbamate23 (30 mg, 0.0687 mmol) in CH₂Cl₂ (1.5 mL) at 0° C. was added TFA (0.5mL). The reaction mixture was stirred at 0° C. for 2.0 h andconcentrated. The crude amine was used for the next reaction withoutfurther purification.

To a solution of crude amine and 3,3,3-triphenyl propionic acid (20 mg,0.068 mmol) in CH₂Cl₂ (1.0 mL) at rt were added HOBt (11 mg, 0.0816mmol) and DIEA (36 μL, 0.204 mmol). The mixture was cooled to 0° C. andEDCI (16 mg, 0.0816 mmol) was added to the mixture. The resultingmixture was allowed to rt, stirred at rt for 17 h, and quenched at 0° C.with H₂O (3 mL). The mixture was extracted three times with ethylacetate and the combined extracts were washed with brine, dried overNa₂SO₄, filtered, and concentrated. The residue was chromatographed onsilica gel to provide 24 (HyT9, 35 mg, 83%). ¹H NMR (500 MHz, CD₃OD) δ7.89 (s, 1H), 7.23-7.16 (m, 13H), 7.12-7.09 (m, 3H), 3.55-3.45 (m, 10H),3.42 (t, J=6.5 Hz, 2H), 3.30-3.28 (m, 2H), 2.80-2.78 (m, 2H), 2.07 (t,J=7.2 Hz, 2H), 1.73-1.67 (m, 2H), 1.56-1.50 (m, 2H), 1.45-1.37 (m, 4H),1.36-1.31 (m, 2H), 1.11-1.09 (m, 2H), 1.05-1.02 (m, 2H). ¹³C NMR (125MHz, CD₃OD) δ 176.1, 172.9, 172.8, 148.3, 130.6, 128.6, 127.1, 72.2,71.2, 71.1, 70.6, 57.6, 48.2, 40.3, 40.0, 36.8, 33.7, 30.5, 29.7, 27.7,27.4, 26.5, 26.4. HRMS (ES+) calculated for C₃₇H₅₀N₂O₄Cl [M+H]⁺621.3459. found 621.3460. TLC (10% CH₃OH in CH₂Cl₂), R_(f) 0.48 (UV,CAM).

N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)-3,3,3-triphenylpropanamide(HyT10, 25)

HyT10 was synthesized by the similar methods as HyT9.

¹H NMR (400 MHz, CDCl₃) δ 7.29-7.17 (m, 15H), 7.12 (s, 1H), 5.68 (s,1H), 4.00 (s, 2H), 3.66-3.64 (m, 2H), 3.62-3.60 (m, 2H), 3.59-3.57 (m,4H), 3.55-3.51 (m, 8H), 3.20-3.17 (m, 2H), 3.15-3.13 (m, 2H), 1.80-1.73(m, 2H), 1.62-1.55 (m, 2H), 1.49-1.41 (m, 2H), 1.39-1.32 (m, 2H). ¹³CNMR (100 MHz, CDCl₃) δ 170.5, 170.1, 146.5, 129.2, 127.9, 126.2, 71.2,70.6, 70.5, 70.4, 70.3, 70.2, 69.9, 69.7, 69.5, 56.1, 48.1, 45.0, 39.0,38.5, 32.4, 29.4, 26.6, 25.3. HRMS (ES+) calculated for C₃₉H₅₄N₂O₇Cl[M+H]⁺ 697.3620. found 697.3622. TLC (10% CH₃OH in CH₂Cl₂), R_(f) 0.43(UV, CAM).

(S)—N-(2-(2-((6-Chlorohexyl)oxy)ethoxy)ethyl)-2-(3,4,5-trimethoxyphenyl)butanamide(HyT11, 26)

HyT11 was synthesized by the same methods as HyT13 (3).

¹H NMR (400 MHz, CDCl₃) δ 6.51 (s, 2H), 6.01 (brs, 1H), 3.84 (s, 6H),3.81 (s, 3H), 3.54-3.45 (m, 9H), 3.43-3.32 (m, 3H), 3.10 (t, J=7.5 Hz,1H), 2.17-2.06 (m, 1H), 1.85-1.70 (m, 3H), 1.61-1.54 (m, 2H), 1.47-1.39(m, 2H), 1.37-1.30 (m, 2H), 0.87 (t, J=7.3 Hz, 3H). ¹³C NMR (100 MHz,CDCl₃) δ 173.3, 153.2, 136.8, 135.8, 104.7, 71.1, 70.2, 69.9, 69.8,60.7, 56.0, 55.3, 44.9, 39.2, 32.4, 29.3, 26.6, 26.5, 25.3, 12.3. HRMS(ES+) calculated for C₂₃H₃₉NO₆Cl [M+H]⁺ 460.2466. found 460.2465. TLC(10% CH₃OH in CH₂Cl₂), R_(f) 0.62 (UV, CAM).

2-(4-((3r,5r,7r)-Adamantan-1-yl)phenoxy)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)acetamide (HyT14, 27)

HyT14 was synthesized by the same methods as HyT13 (3).

To a solution of (4-adamantan-1-yl-phenoxy) acetic acid 19 (27 mg, 0.094mmol) and amine 8 (21 mg, 0.094 mmol) in CH₂Cl₂ (1.5 mL) at rt wereadded HOBt (15 mg, 0.113 mmol) and DIEA (50 μL, 0.282 mmol). Thereaction mixture was cooled to 0° C. and EDCI (22 mg, 0.113 mmol) wasadded to the mixture. The resulting mixture was stirred at rt for 22 hand quenched at 0° C. with H₂O (4 mL). The mixture was extracted twicewith ethyl acetate and the combined extracts were washed with brine,dried over Na₂SO₄, filtered, and concentrated. The residue waschromatographed on silica gel to afford 27 (HyT14, 42 mg, 92%). ¹H NMR(400 MHz, CDCl₃) δ 7.29 (d, J=8.8 Hz, 2H), 7.04 (brs, 1H), 6.86 (d,J=8.8 Hz, 2H), 4.47 (s, 2H), 3.58-3.52 (m, 8H), 3.51 (t, J=6.7 Hz, 2H),3.44 (d, J=6.7 Hz, 2H), 2.08 (s, 3H), 1.87 (s, 6H), 1.79-1.71 (m, 8H),1.62-1.55 (m, 2H), 1.46-1.38 (m, 2H), 1.37-1.30 (m, 2H). ¹³C NMR (100MHz, CDCl₃) δ 168.4, 155.0, 145.1, 126.0, 114.2, 71.2, 70.3, 69.9, 67.4,45.0, 43.3, 38.7, 36.7, 35.6, 32.4, 29.4, 28.8, 26.6, 25.3. HRMS (ES+)calculated for C₂₈H₄₃NO₄Cl [M+H]⁺ 492.2881. found 492.2883. TLC (10%CH₃OH in CH₂Cl₂), R_(f) 0.65 (UV, CAM).

(1S)—N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-carboxamide(HyT15, 28)

Hy15 was synthesized by the same methods as HyT13 (3).

¹H NMR (400 MHz, CDCl₃) δ 7.79 (s, 1H), 3.62-3.53 (m, 6H), 3.54-3.48 (m,4H), 3.46 (t, J=6.4 Hz, 2H), 2.53 (dd, J=13.9, 4.0 Hz, 1H), 2.48 (dd,J=5.6, 5.0 Hz, 1H), 2.17-2.09 (m, 1H), 2.07 (t, J=4.5 Hz, 1H), 1.95 (d,J=18.6 Hz, 2H), 1.80-1.73 (m, 2H), 1.62-1.55 (m, 2H), 1.47-1.32 (m, 4H).¹³C NMR (100 MHz, CDCl₃) δ 216.9, 169.1, 71.2, 70.4, 70.0, 69.8, 64.6,50.1, 45.0, 43.7, 43.2, 38.6, 32.5, 29.4, 28.1, 27.6, 26.7, 25.4, 20.9,20.4. HRMS (ES+) calculated for C₂₀H₃₅NO₄Cl [M+H]⁺ 388.2255. found388.2253. TLC (10% CH₃OH in CH₂Cl₂), R_(f) 0.57 (UV, CAM).

N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-6-fluoro-2-naphthamide (HyT17,29)

HyT17 was synthesized by the same methods as HyT13 (3).

¹H NMR (400 MHz, CDCl₃) δ 8.31 (s, 1H), 7.91 (dd, J=9.0, 5.6 Hz, 1H),7.85 (dd, J=8.7, 8.7 Hz, 1H), 7.83 (dd, J=8.5, 8.5 Hz, 1H), 7.47 (dd,J=9.6, 2.4 Hz, 1H), 7.31 (ddd, J=8.7, 8.7, 2.5 Hz, 1H), 3.72-3.70 (m,4H), 3.69-3.66 (m, 2H), 3.62-3.59 (m, 2H), 3.46 (d, J=6.5 Hz, 2H), 3.44(d, J=6.5 Hz, 2H), 1.71-1.64 (m, 2H), 1.57-1.50 (m, 2H), 1.39-1.21 (m,4H). ¹³C NMR (100 MHz, CDCl₃) δ 167.2, 162.8, 160.3, 135.6, 135.5,131.4, 131.3, 131.23, 131.21, 129.5, 127.7, 127.6, 127.5, 124.6, 117.3,117.1, 111.0, 110.8, 71.2, 70.2, 69.9, 69.7, 44.9, 39.7, 32.4, 29.4,26.5, 25.3. HRMS (ES+) calculated for C₂₁H₂₈NO₃ClF [M+H]⁺ 396.1742.found 396.1744. TLC (10% CH₃OH in CH₂Cl₂), R_(f) 0.65 (UV, CAM).

2-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)-N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)acetamide (HyT18, 30)

HyT18 was synthesized by the same methods as HyT9.

¹H NMR (400 MHz, CD₃OD) δ 7.29 (d, J=8.8 Hz, 2H), 6.90 (d, J=8.8 Hz,2H), 4.47 (s, 2H), 3.95 (s, 2H), 3.64 (s, 4H), 3.60-3.51 (m, 14H),3.47-3.39 (m, 6H), 2.06 (s, 3H), 1.89 (d, J=2.2 Hz, 6H), 1.80-1.70 (m,8H), 1.59-1.52 (m, 2H), 1.48-1.42 (m, 2H), 1.41-1.34 (m, 2H). ¹³C NMR(100 MHz, CD₃OD) δ 172.8, 171.4, 156.9, 146.1, 127.0, 115.4, 72.2, 71.9,71.5, 71.4, 71.3, 71.2, 71.1, 70.5, 70.4, 68.4, 45.7, 44.5, 39.9, 39.8,37.8, 36.7, 33.7, 30.5, 30.4, 27.7, 26.5. HRMS (ES+) calculated forC₃₆H₅₈N₂O₈Cl [M+H]⁺ 681.3871. found 681.3861. TLC (10% CH₃OH in CH₂Cl₂),R_(f) 0.62 (UV, CAM).

N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-(((2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)acetamide (HyT23, 31)

HyT23 was synthesized by the same methods as 3.

¹H NMR (400 MHz, CDCl₃) δ 6.97 (brs, 1H), 4.05 (d, J=15.1 Hz, 1H), 3.84(d, J=15.1 Hz, 1H), 3.61-3.59 (m, 2H), 3.57-3.54 (m, 4H), 3.52 (t, J=6.7Hz, 2H), 3.49 (t, J=5.0 Hz, 2H), 3.45 (t, J=6.6 Hz, 2H), 3.13 (td,J=10.6, 4.1 Hz, 1H), 2.12 (dtd, J=14.0, 7.0, 2.8 Hz, 1H), 2.06-2.00 (m,1H), 1.80-1.73 (m, 2H), 1.67-1.63 (m, 2H), 1.62-1.55 (m, 2H), 1.48-1.23(m, 7H), 0.96 (qd, J=13.8, 3.2 Hz, 1H), 0.91 (d, J=1.2 Hz, 3H), 0.90 (d,J=1.8 Hz, 3H), 0.89-0.81 (m, 2H), 0.77 (d, J=6.9 Hz, 3H). ¹³C NMR (100MHz, CDCl₃) δ 170.4, 80.5, 71.2, 70.3, 70.0, 69.8, 67.9, 47.9, 45.0,40.1, 38.4, 34.3, 32.5, 31.3, 29.4, 26.6, 25.9, 25.4, 23.2, 22.2, 20.9,16.2. HRMS (ES+) calculated for C₂₂H₄₃NO₄Cl [M+H]⁺ 420.2881. found420.2881. TLC (33% EtOAc in Hexanes), R_(f) 0.14 (CAM).

(R)—N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-(2-fluoro-[1,1′-biphenyl]-4-yl)propanamide (HyT24, 32)

HyT24 was synthesized by the same methods as HyT13 (3).

¹H NMR (400 MHz, CDCl₃) δ 7.53-7.51 (m, 2H), 7.45-7.33 (m, 4H),7.17-7.11 (m, 2H), 6.04 (brs, 1H), 3.59-3.48 (m, 9H), 3.46-3.39 (m, 4H),1.78-1.71 (m, 2H), 1.60-1.53 (m, 2H), 1.54 (d, J=7.1 Hz, 3H), 1.46-1.39(m, 2H), 1.37-1.29 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 173.4, 160.9,158.4, 142.9, 142.8, 135.4, 130.9, 130.8, 128.9, 128.8, 128.4, 127.7,127.6, 71.2, 70.2, 69.9, 69.6, 46.5, 44.9, 39.3, 32.4, 29.4, 26.6, 25.3,18.5. HRMS (ES+) calculated for C₂₅H₃₄NO₃ClF [M+H]⁺ 450.2211. found450.2209. TLC (10% CH₃OH in CH₂Cl₂), R_(f) 0.66 (UV, CAM).

2-(2,2,4,7-tetramethyl-3,4-dihydroquinolin-1(2H)-yl)ethyl(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl) carbamate (HyT25, 33)

HyT25 was synthesized by the similar methods as 3.

¹H NMR (400 MHz, CD₃OD) δ 6.97 (d, J=7.7 Hz, 1H), 6.51 (s, 1H), 6.42 (d,J=7.7 Hz, 1H), 4.13 (dd, J=8.7, 5.8 Hz, 1H), 4.06 (dd, J=8.7, 5.7 Hz,1H), 3.64-3.56 (m, 4H), 3.55-3.50 (m, 4H), 3.47 (t, J=6.5 Hz, 2H),3.30-3.26 (m, 4H), 2.87-2.78 (m, 1H), 2.23 (s, 3H), 1.77-1.70 (m, 3H),1.62-1.55 (m, 2H), 1.48-1.35 (m, 5H), 1.29 (s, 3H), 1.26 (d, J=6.7 Hz,3H), 1.13 (s, 3H). ¹³C NMR (125 MHz, CD₃OD) δ 159.0, 145.8, 137.3,126.9, 126.5, 118.1, 113.4, 72.2, 71.3, 71.2, 70.9, 63.8, 55.2, 45.6,44.9, 41.7, 33.7, 30.5, 30.0, 28.3, 27.7, 26.5, 24.4, 21.8, 20.8. HRMS(ES+) calculated for C₂₆H₄₄N₂O₄Cl [M+H]⁺ 483.2990. found 483.2986. TLC(10% CH₃OH in CH₂Cl₂), R_(f) 0.43 (UV, CAM).

2-(10H-phenoxazin-10-yl)ethyl(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)carbamate (HyT26, 34)

HyT26 was synthesized by the similar methods as HyT13 (3).

¹H NMR (500 MHz, CD₃OD) δ 6.79 (dd, J=8.2, 1.5 Hz, 1H), 6.78 (dd, J=8.2,1.5 Hz, 1H), 6.72 (d, J=7.5 Hz, 2H), 6.64 (dd, J=7.5, 1.3 Hz, 1H), 6.62(dd, J=7.4, 1.3 Hz, 1H), 6.57 (dd, J=7.8, 1.3 Hz, 2H), 4.25 (t, J=6.4Hz, 2H), 3.81 (t, J=6.4 Hz, 2H), 3.56-3.53 (m, 3H), 3.51 (t, J=6.6 Hz,3H), 3.48 (t, J=5.5 Hz, 2H), 3.44 (t, J=6.5 Hz, 2H), 3.26 (t, J=5.5 Hz,2H), 1.76-1.70 (m, 2H), 1.59-1.53 (m, 2H), 1.46-1.40 (m, 2H), 1.39-1.32(m, 2H). ¹³C NMR (125 MHz, CD₃OD) δ 159.1, 146.5, 134.8, 125.3, 122.6,116.7, 113.5, 72.6, 71.7, 71.6, 71.3, 61.6, 46.1, 44.7, 42.2, 34.1,30.9, 28.2, 26.9. HRMS (ES+) calculated for C₂₅H₃₄N₂O₅Cl [M+H]⁺477.2156. found 477.2152. TLC (10% CH₃OH in CH₂Cl₂), R_(f) 0.43 (UV,CAM).

(R)—N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3,3,3-trifluoro-2-methoxy-2-phenylpropanamide (HyT27, 35)

HyT27 was synthesized by the same methods as HyT13 (3).

¹H NMR (400 MHz, CDCl₃) δ 7.55-7.53 (m, 2H), 7.40-7.38 (m, 3H), 7.17(brs, 1H), 3.59-3.56 (m, 5H), 3.54-3.48 (m, 5H), 3.43 (t, J=6.7 Hz, 2H),3.41 (s, 3H), 1.79-1.72 (m, 2H), 1.61-1.54 (m, 2H), 1.48-1.40 (m, 2H),1.39-1.31 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 166.3, 132.6, 129.4,128.4, 127.6, 71.2, 70.2, 69.9, 69.4, 54.9, 45.0, 39.1, 32.4, 29.4,26.6, 25.3. HRMS (ES+) calculated for C₂₀H₃₀NO₄ClF₃ [M+H]⁺ 440.1815.found 440.1814. TLC (33% EtOAc in Hexanes), R_(f) 0.51 (UV, CAM).

(R)-1-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-3-(3-methyl-1,1-diphenylbutan-2-yl)urea(HyT29, 36)

HyT29 was synthesized by the same methods as HyT13 (3).

¹H NMR (400 MHz, CDCl₃) δ 7.38 (dd, J=6.7, 6.7 Hz, 4H), 7.32-7.25 (m,4H), 7.22-7.14 (m, 2H), 4.72 (t, J=9.7 Hz, 1H), 4.60 (t, J=5.7 Hz, 1H),4.23 (d, J=9.8 Hz, 1H), 3.95 (d, J=11.0 Hz, 1H), 3.57 (t, J=6.6 Hz, 2H),3.55-3.42 (m, 7H), 3.37-3.32 (m, 1H), 3.30-3.15 (m, 2H), 1.85-1.78 (m,2H), 1.76-1.68 (m, 1H), 1.67-1.60 (m, 2H), 1.54-1.46 (m, 2H), 1.44-1.36(m, 2H), 0.98 (d, J=6.8 Hz, 3H), 0.88 (d, J=6.8 Hz, 3H), 0.04 (s, 6H).¹³C NMR (100 MHz, CDCl₃) δ 158.3, 143.1, 142.8, 128.7, 128.4, 128.3,127.9, 126.4, 126.3, 71.2, 70.9, 70.3, 70.0, 55.7, 45.0, 40.5, 32.5,29.4, 29.2, 26.6, 25.4, 20.8, 15.1, 0.0. HRMS (ES+) calculated forC₂₈H₄₂N₂O₃Cl [M+H]⁺ 489.2884. found 489.2881. TLC (10% CH₃OH in CH₂Cl₂),R_(f) 0.62 (UV, CAM).

2-(bis((R)-1-phenylethyl)amino)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)acetamide(HyT30, 37)

HyT30 was synthesized by the same methods as HyT13 (3).

¹H NMR (500 MHz, CD₃OD) δ 7.34 (s, 4H), 7.33 (s, 4H), 7.27-7.23 (m, 2H),4.87 (s, 2H), 3.95 (q, J=6.8 Hz, 2H), 3.67-3.60 (m, 4H), 3.52-3.42 (m,6H), 3.38 (d, J=17.6 Hz, 1H), 3.27-3.16 (m, 2H), 2.85 (d, J=17.6 Hz,1H), 1.73-1.68 (m, 2H), 1.60-1.54 (m, 2H), 1.45-1.34 (m, 4H), 1.35 (d,J=6.8 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 170.0, 142.7, 128.4, 127.7,127.2, 71.3, 70.4, 70.0, 69.8, 59.5, 50.3, 45.0, 38.4, 32.4, 29.4, 26.5,25.4, 20.0, −0.03. HRMS (ES+) calculated for C₂₈H₄₂N₂O₃Cl [M+H]⁺489.2884. found 489.2883. TLC (10% CH₃OH in CH₂Cl₂), R_(f) 0.56 (UV,CAM).

Ketone 38 and Alcohol 39 were prepared by the reported procedure(Bioorg. Med. Chem., 1998, 6, 1309-1335).

Ketone 38:

¹H NMR (500 MHz, CDCl₃) δ 7.55 (d, J=7.9 Hz, 1H), 7.44 (dd, J=2.6, 1.4Hz, 1H), 7.35 (t, J=7.9 Hz, 1H), 7.10 (ddd, J=8.2, 2.7, 0.8 Hz, 1H),6.79-6.74 (m, 3H), 4.54 (s, 2H), 3.85 (s, 3H), 3.83 (s, 3H), 3.23 (t,J=8.0 Hz, 2H), 2.98 (t, J=6.9 Hz, 2H), 1.47 (s, 9H). ¹³C NMR (125 MHz,CDCl₃) δ 198.8, 167.6, 158.1, 148.9, 147.4, 138.2, 133.8, 129.7, 121.4,120.1, 120.0, 113.1, 111.8, 111.3, 82.6, 77.6, 65.6, 55.9, 55.8, 40.7,29.8, 28.0.

Alcohol 39:

¹H NMR (500 MHz, CDCl₃) δ 7.26 (dd, J=8.2, 8.2 Hz, 1H), 6.96 (d, J=7.7Hz, 1H), 6.93 (s, 1H), 6.81-6.78 (m, 2H), 6.74-6.71 (m, 2H), 4.68-4.65(m, 1H), 4.52 (s, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 2.72-2.66 (m, 1H),2.64-2.58 (m, 1H), 2.12-2.04 (m, 1H), 2.02-1.95 (m, 1H), 1.83 (d, J=3.3Hz, 1H), 1.55 (s, 1H), 1.48 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 168.0,158.1, 148.8, 147.2, 146.4, 134.3, 129.6, 120.2, 119.1, 113.6, 112.2,111.7, 111.2, 82.4, 77.2, 73.7, 65.6, 55.9, 55.8, 40.6, 31.6, 28.0. TLC(33% EtOAc in Hexanes), R_(f) 0.19 (UV, CAM).

tert-Butyl2-(3-((S)-1-(4-((3S,5S,7S)-adamantan-1-yl)phenoxy)-3-(3,4-dimethoxyphenyl)propyl) phenoxy)acetate 40

To a solution of alcohol 39 (48 mg, 0.1193 mmol), 4-(1-adamantyl)phenol(27 mg, 0.1193 mmol), and triphenylphosphine (35 mg, 0.1312 mmol) in THF(1.2 mL) at rt was added DIAD (26 μL, 0.1312 mmol). The resultingmixture was stirred at rt for 20 h, and diluted at rt with H₂O/EtOAc(1:1, 5 mL). The mixture was extracted twice with ethyl acetate and thecombined extracts were washed with brine, dried over Na₂SO₄, filtered,and concentrated. The residue was chromatographed on silica gel toprovide tert-Butyl2-(3-((S)-1-(4-((3S,5S,7S)-adamantan-1-yl)phenoxy)-3-(3,4-dimethoxyphenyl)propyl) phenoxy)acetate 40 (55 mg, 76%). ¹H NMR (400 MHz, CDCl₃) δ 7.23(dd, J=7.9, 7.9 Hz, 1H), 7.15 (d, J=8.8 Hz, 2H), 6.95 (d, J=7.7 Hz, 1H),6.88 (s, 1H), 6.80-6.75 (m, 4H), 6.72 (ddd, J=8.2, 8.2, 1.8 Hz, 1H),6.62 (d, J=1.6 Hz, 1H), 4.94 (dd, J=8.9, 4.0 Hz, 1H), 4.48 (s, 2H), 3.85(s, 3H), 3.65 (s, 3H), 2.80-2.72 (m, 2H), 2.29-2.20 (m, 1H), 2.05-1.96(m, 4H), 1.82-1.69 (m, 11H), 1.45 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ167.9, 158.1, 156.0, 148.5, 147.0, 144.3, 143.6, 133.9, 129.6, 125.6,120.1, 119.0, 115.1, 113.2, 112.0, 111.9, 111.1, 82.3, 78.5, 77.2, 65.5,55.8, 55.4, 43.2, 40.6, 36.7, 35.4, 31.5, 28.9, 27.9. LRMS (ES+)[M+Na]⁺635.8, TLC (25% EtOAc in Hexanes), R_(f) 0.57 (UV, CAM).

2-(3-((S)-1-(4-((3S,5S,7S)-adamantan-1-yl)phenoxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid 41

To a stirred solution of tert-Butyl2-(3-((S)-1-(4-((3S,5S,7S)-adamantan-1-yl)phenoxy)-3-(3,4-dimethoxyphenyl)propyl) phenoxy)acetate 40 (40 mg, 0.0653 mmol) in CH₂Cl₂ (2.0 mL) at 0°C. was added TFA (0.15 mL). The reaction mixture was stirred at 0° C.for 2.0 h and concentrated. The residue was chromatographed on silicagel to provide acid 41 (31 mg, 85%). ¹H NMR (400 MHz, CD₃OD) δ 7.22 (dd,J=7.9, 7.9 Hz, 1H), 7.13 (d, J=8.8 Hz, 2H), 6.93-6.88 (m, 2H), 6.84 (d,J=8.1 Hz, 1H), 6.78 (dd, J=8.2, 2.2 Hz, 1H), 6.74 (d, J=8.8 Hz, 2H),6.71 (dd, J=8.2, 1.7 Hz, 1H), 6.67 (d, J=1.7 Hz, 1H), 4.97 (dd, J=8.8,4.2 Hz, 1H), 4.59 (s, 2H), 3.78 (s, 3H), 3.56 (s, 3H), 2.74 (t, J=7.6Hz, 2H), 2.22-2.15 (m, 1H), 2.03-1.96 (m, 4H), 1.84-1.79 (m, 6H),1.78-1.71 (m, 6H). ¹³C NMR (100 MHz, CD₃OD) δ 159.7, 157.4, 150.2,148.6, 145.7, 144.9, 135.6, 130.7, 126.6, 121.7, 120.2, 116.4, 114.3,113.7, 113.5, 113.2, 79.4, 56.5, 56.1, 44.5, 41.8, 37.8, 36.6, 32.5,30.5. TLC (10% CH₃OH in CH₂Cl₂), R_(f) 0.24 (UV, CAM).

2-(3-4S)-1-(443S,5S,7S)-adamantan-1-yl)phenoxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)-N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)acetamide(HyT31, 42)

42 synthesized by the similar methods as 3. ¹H NMR (500 MHz, CD₃OD) δ7.25 (dd, J=7.1, 7.1 Hz, 1H), 7.15 (d, J=8.7 Hz, 2H), 6.96 (d, J=6.8 Hz,2H), 6.84 (d, J=8.2 Hz, 2H), 6.75 (d, J=8.8 Hz, 2H), 6.71 (d, J=8.2 Hz,1H), 6.67 (d, J=1.4 Hz, 1H), 5.00 (dd, J=8.9, 4.0 Hz, 1H), 4.47 (d,J=3.2 Hz, 2H), 3.92 (s, 2H), 3.77 (s, 3H), 3.59 (s, 3H), 3.56-3.50 (m,18H), 3.43-3.36 (m, 6H), 2.76 (t, J=7.0 Hz, 2H), 2.25-2.17 (m, 1H),2.02-1.97 (m, 4H), 1.84-1.69 (m, 14H), 1.56-1.51 (m, 2H), 1.45-1.39 (m,2H), 1.37-1.32 (m, 2H). ¹³C NMR (125 MHz, CD₃OD) δ 172.7, 171.1, 159.5,157.4, 150.4, 148.8, 145.9, 145.0, 135.8, 130.9, 126.7, 121.8, 120.7,116.5, 114.8, 113.7, 113.5, 79.5, 72.2, 72.0, 71.4, 71.33, 71.31, 71.2,71.1, 70.5, 70.4, 68.4, 56.7, 56.3, 45.7, 44.6, 41.8, 40.0, 39.8, 37.9,36.6, 33.7, 32.5, 30.6, 30.5, 27.7, 26.5. HRMS (ES+) calculated forC₅₃H₇₆N₂O₁₁Cl [M+H]⁺ 951.5138. found 951.5142. TLC (10% CH₃OH inCH₂Cl₂), R_(f) 0.68 (UV, CAM).

N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-4-pentylbicyclo[2.2.2]octane-1-carboxamide(HyT33, 43)

HyT33 was synthesized by the same methods as HyT13 (3).

¹H NMR (400 MHz, CDCl₃) δ 6.01 (t, J=4.8 Hz, 1H), 3.60-3.58 (m, 2H),3.56-3.54 (m, 2H), 3.52 (t, J=6.5 Hz, 4H), 3.45 (t, J=6.7 Hz, 2H), 3.19(t, J=5.3 Hz, 2H), 1.80-1.73 (m, 2H), 1.72-1.68 (m, 6H), 1.63-1.56 (m,2H), 1.49-1.41 (m, 2H), 1.40-1.34 (m, 8H), 1.30-1.23 (m, 2H), 1.20-1.12(m, 4H), 1.09-1.03 (m, 2H), 0.86 (t, J=7.0 Hz, 3H). ¹³C NMR (100 MHz,CDCl₃) δ 178.2, 77.2, 71.2, 70.2, 69.9, 69.8, 45.0, 41.2, 39.0, 38.9,32.7, 32.5, 30.6, 30.3, 29.4, 28.8, 26.6, 25.4, 23.3, 22.6, 14.0. HRMS(ES+) calculated for C₂₄H₄₅NO₃Cl [M+H]⁺ 430.3088. found 430.3088. TLC(5% CH₃OH in CH₂Cl₂), R_(f) 0.51 (CAM).

To a solution of amine 8 (23 mg, 0.1 mmol) in CH₂Cl₂ (1.5 mL) at rt weretriethylamine (140 μL, 1.0 mmol) and 1-adamantyl isocyanate (18 mg, 0.1mmol). The reaction mixture was stirred at rt for 16 h, and evaporated.The residue was chromatographed on silica gel to give1-((3s,5s,7s)-adamantan-1-yl)-3-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)urea44 (HyT34, 40 mg, 76%). ¹H NMR (400 MHz, CDCl₃) δ 4.81 (t, J=5.6 Hz,1H), 4.46 (s, 1H), 3.60-3.58 (m, 2H), 3.56-3.54 (m, 2H), 3.53 (t, J=6.7Hz, 2H), 3.52 (t, J=6.7 Hz, 2H), 3.44 (t, J=6.7 Hz, 2H), 3.31 (t, J=5.4Hz, 1H), 3.30 (t, J=5.4 Hz, 1H), 2.04 (brs, 3H), 1.93 (t, J=2.8 Hz, 6H),1.79-1.72 (m, 2H), 1.64 (t, J=2.8 Hz, 6H), 1.62-1.56 (m, 2H), 1.48-1.40(m, 2H), 1.39-1.31 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 157.4, 77.2,71.2, 70.9, 70.2, 70.0, 50.7, 45.0, 42.4, 39.9, 36.4, 32.4, 29.5, 29.4,26.6, 25.3. HRMS (ES+) calculated for C₂₁H₃₈N₂O₃Cl [M+H]⁺ 401.2571.found 401.2573. TLC (10% CH₃OH in CH₂Cl₂), R_(f) 0.59 (CAM).

1-((3s,5s,7s)-Adamantan-1-yl)-3-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)thiourea(HyT35, 45)

HyT35 was synthesized by the same methods as HyT34 (44).

¹H NMR (500 MHz, CDCl₃) δ 6.20 (s, 1H), 3.76 (s, 2H), 3.63-3.60 (m, 3H),3.55-3.49 (m, 5H), 3.45-3.38 (m, 2H), 2.14-1.93 (m, 9H), 1.77-1.72 (m,2H), 1.69-1.63 (m, 6H), 1.59-1.54 (m, 2H), 1.48-1.39 (m, 2H), 1.38-1.30(m, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 180.8, 77.2, 71.3, 70.4, 70.1, 53.8,45.0, 42.4, 42.1, 42.0, 36.2, 32.5, 29.7, 29.6, 29.5, 26.8, 25.4. HRMS(ES+) calculated for C₂₁H₃₈N₂O₂SCl [M+H]⁺ 417.2343. found 417.2341. TLC(10% CH₃OH in CH₂Cl₂), R_(f) 0.58 (CAM).

(3r,5r,7r)-1-(2-iodoethyl)adamantine 46

To a stirred solution of PPh₃ (1.57 g, 6.0 mmol) in CH₂Cl₂ (14 mL) at rtwere added imidazole (442 mg, 6.5 mmol) and iodine (1.52 g, 6.0 mmol).The reaction mixture was cooled to 0° C. and stirred at 0° C. for 5 min.A solution of 1-adamantane ethanol (901 mg, 5.0 mmol) in CH₂Cl₂ (6 mL)was added dropwise to the mixture via cannula. The resulting mixture wasstirred at 0° C. for 2.0 h and H₂O (20 mL) was added to the mixture atice-bath. The organic layer was separated and the aqueous phase wasextracted twice with CH₂Cl₂. The combined organic layers wereconcentrated. The concentrate was purified by column chromatography toafford (3r,5r,7r)-1-(2-iodoethyl)adamantine 46 (1.375 g, 95%) as a whitesolid. ¹H NMR (400 MHz, CDCl₃) δ 3.17 (d, J=17.9 Hz, 1H), 3.17 (dt,J=3.2, 1.8 Hz, 1H), 1.95 (brs, 3H), 1.78 (d, J=17.9 Hz, 1H), 1.78 (dt,J=3.3, 1.8 Hz, 1H), 1.71 (brs, 1H), 1.68 (brs, 2H), 1.63-1.61 (m, 1H),1.61-1.58 (m, 1H), 1.49 (d, J=2.4 Hz, 1H). TLC (10% EtOAc in Hexanes),R_(f) 0.81 (UV, CAM).

(R)-4-((3R,5R,7R)-adamantan-1-yl)-N-((1S,2S)-1-hydroxy-1-phenylpropan-2-yl)-N,2-dimethylbutanamide 47

A solution of n-butyllithium (2.5 M in hexanes, 0.8 mL, 2.0 mmol, 4.0eq.) was added to a suspension of lithium chloride (275 mg, 6.5 mmol,13.0 eq.) and diisopropylamine (0.3 mL, 2.15 mmol, 4.3 eq.) in THF (2mL) at −78° C. The resulting suspension was warmed briefly to 0° C.,then was cooled to −78° C. An ice-cooled solution of(1S,2S)-(+)-pseudoephedrine propionamide (221 mg, 1.0 mmol, 2.0 eq.) inTHF (2 mL) was added dropwise over 30 min via cannula and the reactionmixture was stirred at 78° C. for 1.0 h, at 0° C. for 15 min, and atroom temperature for 5 min, and cooled to 0° C. To this solution at 0°C. was added a solution of iodide 46 (145 mg, 0.5 mmol, 1.0 eq.) in THF(1 mL) via cannula, and the reaction mixture was stirred at 0° C. for 6h and at room temperature for 20 h. The pale yellow mixture was cooledto 0° C., then treated with half-saturated aqueous NH₄Cl solution (10mL), and extracted with ethyl acetate (10 mL×3). The combined organiclayers were dried over Na₂SO₄, filtered, and concentrated. The residuewas purified by flash chromatography on silica gel to afford(R)-4-((3R,5R,7R)-adamantan-1-yl)-N-((1S,2S)-1-hydroxy-1-phenylpropan-2-yl)-N,2-dimethylbutanamide47 (178 mg, 93%). ¹H NMR (400 MHz, CDCl₃) δ 7.38-7.30 (m, 5H), 4.62 (dd,J=7.3, 7.3 Hz, 1H), 4.35 (brs, 1H), 2.81 (s, 3H), 2.50-2.41 (m, 1H),1.93 (s, 3H), 1.71-1.60 (m, 7H), 1.57-1.41 (m, 8H), 1.17 (d, J=7.0 Hz,3H), 1.08 (d, J=6.7 Hz, 3H), 0.98-0.87 (m, 2H). ¹³C NMR (100 MHz, CDCl₃)δ 179.3, 142.7, 128.7, 128.3, 127.5, 126.9, 126.3, 77.2, 76.5, 42.3,42.2, 37.4, 37.2, 32.1, 28.7, 26.9, 17.2, 15.4. TLC (33% EtOAc inHexanes), R_(f) 0.24 (UV, CAM).

(R)-4-((3R,5R,7R)-adamantan-1-yl)-2-methylbutanoic acid 48

To a solution of (R)-4-((3R,5R,7R)-adamantan-1-yl)-N-((1S,2S)-1-hydroxy-1-phenylpropan-2-yl)-N,2-dimethylbutanamide 47 (120 mg,0.313 mmol) in 1,4-dioxane (3 mL) and H₂O (2 mL) at rt was addedn-Bu₄NOH (40% wt % in H₂O, 1.22 mL, 1.878 mmol). The reaction mixturewas stirred at 110° C. for 20 h, cooled to rt, and evaporated. Theresidue was diluted with H₂O (2 mL), cooled to 0° C., adjusted to pH 4with 3N—HCl. The mixture was extracted twice with ethyl acetate and thecombined extracts were washed with brine, dried over Na₂SO₄, filtered,and concentrated. The residue was chromatographed on silica gel toafford acid 48 (73 mg, quant.). ¹H NMR (400 MHz, CDCl₃) δ 10.8 (brs,1H), 2.41-2.32 (m, 1H), 1.93 (brs, 3H), 1.71-1.60 (m, 7H), 1.46 (d,J=2.0 Hz, 6H), 1.42-1.34 (m, 1H), 1.18 (d, J=7.0 Hz, 3H), 1.11-1.03 (m,2H). ¹³C NMR (100 MHz, CDCl₃) δ 183.5, 42.3, 41.7, 40.0, 37.2, 32.1,28.7, 26.5, 16.8. TLC (33% EtOAc in Hexanes), R_(f) 0.62 (CAM).

(R)-4-((3R,5R,7R)-adamantan-1-yl)-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)-2-methylbutanamide (HyT36, 49)

HyT36 was synthesized by the EDC-mediated coupling method as HyT13. ¹HNMR (400 MHz, CDCl₃) δ 5.93 (s, 1H), 3.62-3.59 (m, 2H), 3.57-3.55 (m,2H), 3.54 (t, J=5.0 Hz, 2H), 3.52 (t, J=6.5 Hz, 2H), 3.47-3.43 (m, 4H),2.09-2.01 (m, 1H), 1.92 (s, 3H), 1.79 (t, J=6.7 Hz, 1H), 1.75 (t, J=6.7Hz, 1H), 1.69-1.57 (m, 9H), 1.47-1.27 (m, 11H), 1.12 (d, J=6.8 Hz, 3H),1.03-0.98 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 176.6, 71.2, 70.2, 70.0,44.9, 42.4, 42.2, 42.1, 38.9, 37.2, 32.5, 32.1, 29.4, 28.7, 27.2, 26.6,25.4, 17.8. HRMS (ES+) calculated for C₂₅H₄₅NO₃Cl [M+H]⁺ 442.3088. found442.3086. TLC (5% CH₃OH in CH₂Cl₂), R_(f) 0.40 (CAM).

HyT39 was synthesized by the same methods as HyT36.

N-((1S,2S)-1-hydroxy-1-phenylpropan-2-yl)-N-methyl-3-phenylpropanamide50

To a solution of (1S,2S)-(+)-pseudoephedrine (496 mg, 3.0 mmol) in THF(9 mL) at rt was added triethylamine (0.59 mL, 4.2 mmol). The mixturewas cooled to 0° C., and hydrocinnamoyl chloride (0.54 mL, 3.6 mmol) wasadded to the mixture. The resulting mixture was stirred at 0° C. for 0.5h, quenched with H₂O (10 mL), and extracted twice with ethyl acetate.The combined extracts were washed with brine, dried over Na₂SO₄,filtered, and concentrated. The residue was chromatographed on silicagel to affordN-((1S,2S)-1-hydroxy-1-phenylpropan-2-yl)-N-methyl-3-phenylpropanamide50 (865 mg, 97%). ¹H NMR (500 MHz, CD₃OD) δ 7.38-7.31 (m, 4H), 7.29-7.13(m, 6H), 4.77 (brs, ½ H), 4.58 (dd, J=11.2, 8.2 Hz, 1H), 4.03-3.97 (m, ½H), 2.88 (d, J=10.7 Hz, 3H), 2.87 (t, J=8.1 Hz, 2H), 2.76-2.58 (m, 2H),0.88 (t, J=7.0 Hz, 3H). ¹³C NMR (extra peaks are due to amide-bondrotamers, 125 MHz, CD₃OD) δ 175.7, 175.6, 143.8, 142.6, 142.5, 130.0,129.6, 129.5, 129.4, 129.3, 128.8, 128.1, 128.0, 127.13, 127.11, 76.3,76.1, 59.7, 36.8, 36.2, 32.7, 32.4, 27.9, 15.6, 14.4. TLC (33% EtOAc inHexanes), R_(f) 0.08 (UV, CAM).

(S)-4-((3S,5S,7S)-adamantan-1-yl)-2-benzyl-N-((1S,2S)-1-hydroxy-1-phenylpropan-2-yl)-N-methylbutanamide51

51 was synthesized by the same method as 47. ¹H NMR (400 MHz, CDCl₃) δ7.30-7.28 (m, 4H), 7.27-7.24 (m, 4H), 7.20-7.17 (m, 2H), 4.48 (dd,J=6.5, 6.5 Hz, 1H), 2.80-2.71 (m, 2H), 2.49 (s, 3H), 1.94 (s, 3H),1.72-1.59 (m, 7H), 1.55 (s, 3H), 1.43 (d, J=2.2 Hz, 6H), 0.99 (d, J=7.0Hz, 3H), 0.97-0.92 (m, 1H), 0.90-0.83 (m, 2H). TLC (33% EtOAc inHexanes), R_(f) 0.43 (UV, CAM).

(S)-4-((3S,5S,7S)-adamantan-1-yl)-2-benzylbutanoic acid 52

52 was synthesized by the same method as 48. ¹H NMR (400 MHz, CDCl₃) δ10.15 (brs, 1H), 7.28-7.23 (m, 2H), 7.20-7.14 (m, 3H), 2.94 (dd, J=13.8,8.2 Hz, 1H), 2.76 (dd, J=13.8, 6.5 Hz, 1H), 2.61-2.53 (m, 1H), 1.92 (s,3H), 1.69-1.56 (m, 7H), 1.54-1.46 (m, 1H), 1.43 (d, J=2.0 Hz, 6H),1.13-1.03 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 181.8, 139.2, 128.6,128.4, 126.3, 47.9, 42.3, 41.6, 37.9, 37.2, 32.1, 28.7, 24.8. TLC (25%EtOAc in Hexanes), R_(f) 0.54 (UV).

(S)-4-((3S,5S,7S)-adamantan-1-yl)-2-benzyl-N-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)butanamide(HyT39, 53)

HyT39 was synthesized by the EDC-mediated coupling method as HyT13. ¹HNMR (500 MHz, CDCl₃) δ 7.25-7.22 (m, 2H), 7.17-7.14 (m, 3H), 5.72 (t,J=5.4 Hz, 1H), 3.52 (t, J=6.6 Hz, 2H), 3.50-3.45 (m, 3H), 3.44-3.40 (m,1H), 3.42 (t, J=6.6 Hz, 2H), 3.69-3.33 (m, 2H), 3.31-3.25 (m, 1H),3.23-3.19 (m, 1H), 2.88 (dd, J=13.3, 9.6 Hz, 1H), 2.72 (dd, J=13.4, 5.3Hz, 1H), 2.16-2.11 (m, 1H), 1.92 (s, 3H), 1.79-1.73 (m, 2H), 1.69-1.56(m, 9H), 1.47-1.41 (m, 3H), 1.44 (d, J=2.2 Hz, 6H), 1.38-1.33 (m, 2H),1.09-0.97 (m, 2H). ¹³C NMR (125 MHz, CDCl₃) δ 174.8, 140.1, 128.9,128.2, 126.0, 71.2, 70.1, 70.0, 69.9, 51.0, 44.9, 42.3, 42.1, 39.2,38.8, 37.2, 32.5, 32.1, 29.4, 28.6, 26.6, 25.7, 25.4. HRMS (ES+)calculated for C₃₁H₄₉NO₃Cl [M+H]⁺ 518.3401. found 518.3405. TLC (5%CH₃OH in CH₂Cl₂), R_(f) 0.55 (UV, CAM).

2-((3r,5r,7r)-adamantan-1-yl)-N-(24-chloro-11-oxo-3,6,9,15,18-pentaoxa-12-azatetracosyl)acetamide (HyT40, 54)

HyT40 was synthesized by the similar methods as 3.

¹H NMR (400 MHz, CDCl₃) δ 7.18 (s, 1H), 6.19 (s, 1H), 4.04 (s, 2H),3.70-3.41 (m, 24H), 1.95 (s, 3H), 1.80-1.73 (m, 4H), 1.70-1.55 (m, 8H),1.61 (d, J=2.2 Hz, 6H), 1.48-1.41 (m, 2H), 1.39-1.33 (m, 2H). ¹³C NMR(100 MHz, CDCl₃) δ 171.1, 171.0, 71.2, 70.7, 70.6, 70.5, 70.4, 70.2,70.1, 70.0, 69.9, 69.8, 51.5, 45.0, 42.5, 39.0, 38.6, 36.7, 32.6, 32.5,29.4, 28.6, 26.6, 25.4. HRMS (ES+) calculated for C₃₀H₅₄N₂O₇Cl [M+H]⁺589.3620. found 589.3622. TLC (10% CH₃OH in CH₂Cl₂), R_(f) 0.66 (UV,CAM).

Biological Experiments Methods Cell Culture and Materials

Indicated cells were grown at 37° C. in DMEM, supplemented with 10%fetal bovine serum and penicillin/streptomycin. The HaloTag protein wasobtained from pHT2 vector (Promega). The luciferase sequence wasobtained from pGL3-Basic vector (Promega), mouse Ror2 was kindlyprovided by Sigmar Stricker (Max Planck-Institute for MolecularGenetics), Danio rerio Smad5 was cloned from a zebrafish cDNA libraryand H-RasG12V was obtained from Addgene plasmid 9051, contributed byRobert Weinberg (MIT). The remaining transmembrane proteins were clonedfrom a human spleen cDNA library (Invitrogen). A D106A point mutationwas introduced into the HaloTag gene by the QuikChange Site Directedmutagenesis kit (Stratagene). Flp-In 293 cells were purchased fromInvitrogen. HA-HaloTag-Smad5 and EGFP-HaloTag were cloned into the pCS2+vector, while the rest of the constructs were cloned into a retroviralpEYK3.1 vector (kindly provided by George Daley, MIT) by excising GFP⁴¹.Retrovirus was generated in GP2-293 cells (Clontech) with a pVSV-G and acorresponding pEYK plasmid, and the indicated cells were infected asdescribed⁴¹. Anti-HA antibody was purchased from Covance (clone 16B12)and anti-β-actin antibody was purchased from Sigma (clone AC-74). HyTcompounds were stored and aliquoted in DMSO as 1000× stock solutions.

Luciferase Assay

Ten thousand stable HEK 293T cells infected with HA-luciferase-HaloTagwere plated into each well in a 96-well plate. The next day, indicatedHyT compounds were added in triplicate and the cells were cultured foranother 24 hours. The cells were washed once with cold PBS and lysed inPassive Lysis Buffer (Promega). The luciferase activity was performed bySteady-Glo Luciferase Assay System (Promega) on a Wallac Victor 2 PlateReader (Perkin Elmer) and the luciferase activity was normalized byprotein concentration, as determined by the Bradford assay.

Immunoblotting

The indicated cells were washed twice with cold PBS and the cells werelysed in lysis buffer (lx PBS, 1% NP-40, 1 mM EDTA, 40 mM HEPES) withprotease inhibitors. The lysates were cleared by centrifugation at10,000 g for 5 min. The total protein concentration was determined byBradford assay and 50 μg of protein was loaded onto an 8% Bis-Tris gel.To solubilize polyubiquitinated and aggregated proteins upon proteasomeinhibition⁴² samples generated for FIG. 12d were lysed with a SDS lysisbuffer (lx PBS, 1% NP-40, 1% SDS, 1% sodium deoxycholate, 1 mM EDTA, 40mM HEPES) with protease inhibitors. The blots were processed by standardprocedures with indicated antibodies, and the band intensities werequantified by ImageJ.

Flow Cytometry Analysis

Stable HeLa cells were raised by cotransfection of pCS2/EGFP-HaloTag andp-Puro containing the puromycin resistance gene. A clonal population ofcells expressing EGFP-HaloTag was isolated. These cells were treatedwith vehicle or 1 μM HyT13 for 24 hours, washed with PBS andtrypsinized. The cells were resuspended in FBS-free DMEM and theintracellular GFP level was measured by FACSCalibur (BD Biosciences).

Zebrafish Danio rerio Experiments

The wild-type fish line TLF was used for this study. TheHA-HaloTag-Smad5 in pCS2+ plasmid was in vitro transcribed with the SP6transcription kit (Ambion). The mRNA was injected at 100 ng/μL at theone cell stage and embryos were raised to the 256-cell stage, when theywere moved to glass depression slides (10-per-well) and put in 1 ml E2media with or without HyT13 (10 μM). Embryos were cultured at 28.6° C.for 24 hours and then dechorionated and de-yolked as described⁴³.Approximately 60 embryos per condition were collected for immunoblotanalysis, as described above.

Focus Formation Assay

One hundred thousand NIH-3T3 cells infected with HA-HaloTag-H-RasG12Vand HA-HaloTag(D106A)-H-RasG12V were plated onto 10-cm cell cultureplates in 10% FBS with DMEM. The next day, the media was replaced with1% FBS media and the cells were administered either vehicle or 1 μMHyT13. The media and the drug were replaced every two days. On day 6,the foci were photographed and counted as the number of distinct fociper 1-cm² area.

Tumor Formation Assay

One hundred thousand NIH-3T3 cells expressing HA-HaloTag-H-RasG12V wereinjected into the flank of anesthetized 6-week old female nu/nu nudemice (Charles River Laboratories). Two hours later, the mice were IPinjected with either vehicle (10 μL volume, with 5 μL DMSO and 5 μL ofCremophor EL), 25 mg/kg HyT13 or 100 mg/kg HyT13. The drug injectionscontinued daily until the end of the experiment. Upon the appearance oftumors on day 7, the tumors were measured daily with calipers, and theirvolumes were calculated using the formula: a(b)²/2, where a and brepresent the longest and shortest diameters of the tumor, respectively.

Results Hydrophobic Tagging Destabilizes HaloTag Fusion Proteins

The inventors designed 21 structurally distinct scaffolds as the basisfor our hydrophobic Tags (HyTs), and synthesized and tested 30 compoundsacross these scaffolds composed of hydrophobic moieties linked to theHaloTag haloalkane reactive linker (Table 1, FIG. 5). In designing thehydrophobic portion of these bifunctional molecules, the inventors usedthe compound library available in the Yale University Small MoleculeDiscovery Center as an informal resource to identify compounds that (1)maximized hydrophobicity, (2) minimized molecular weight, and (3)incorporated chemically diverse and commercially available scaffolds. Todetermine their biological activity, we generated a stable HEK 293T cellline expressing a luciferase-HaloTag fusion protein and treated thesecells with the HyT compounds at 1 μM for 24 hours. Remarkably, severalnon-toxic compounds appeared to reduce luciferase activity and wecharacterized the five most potent compounds further (FIG. 1). All fiveHyTs exhibited high hydrophobicity scores (logP ranging from +3 to +5)and were active in a concentration-dependent manner, whereas the HyT5control compound with two PEG groups did not decrease the luciferaseactivity (FIG. 1). Based on these initial data, we continued ourinvestigation of hydrophobic tagging-induced degradation withhydrophobic containing HyT13 because of the reported high stability andcell permeability of compounds bearing adamantyl groups^(27,28).

As the luciferase assay relied on the loss-of-activity of theluciferase-HaloTag fusion protein, we wanted to determine whether thedecrease in luciferase activity resulted from the degradation of theentire fusion protein or perhaps simply inhibition of luciferaseactivity. We generated a stable Flp-In 293 cell line with a singleintegration site containing HA-EGFP-HaloTag fusion protein, and employedthis cell line to perform kinetic studies with HyT13. Immunoblottingshowed that HyT13 efficiently degraded the fusion protein, with amaximal effect achieved at 100 nM (FIG. 12a ). The IC₅₀ of HyT13 wasdetermined to be 21 nM (FIG. 6). A time course experiment revealed thatthe full effect is reached within 8 hours, with 50% degradation observedby 1.5 hours (FIG. 12b and FIG. 7). When cells were treated with 1 μMHyT13 for 24 hours, and then the HyT13 was removed for 24 hours, theprotein level recovered to half the starting levels. No cellulartoxicity was observed at 20 μM of HyT13, a dose of 1000-fold over theIC₅₀ value (FIG. 8). Consistent with our hypothesis that hydrophobictagging mimics a partially denatured protein state and that the proteinis ultimately delivered to the proteasome for degradation, inclusion ofproteasome inhibitors MG132 and YU101²⁹ blocked HyT13 mediateddegradation (FIG. 12c ). To verify that the observed decrease inHA-EGFP-HaloTag levels does not result from masking of the HA epitopeduring immunoblotting, we generated a HeLa cell line stably expressingEGFP-HaloTag and analyzed the intracellular fluorescence by flowcytometry. Consistent with our previous observations, treatment of thesecells with 1 μM of HyT13 for 24 hours reduced the mean fluorescenceintensity of cells almost 7-fold. Together, these findings provide thefirst experimental evidence that hydrophobic tagging represents a viablestrategy for the control of protein levels.

Degradation of Transmembrane and Zebrafish Proteins

One limitation of existing technologies for small molecule control ofprotein levels has been the difficulty of degrading transmembraneproteins′. To determine if hydrophobic tagging shares this limitation,we constructed several transmembrane-HA-HaloTag fusion proteins, suchthat the HaloTag portion would be intracellular. Ror2 is a single-passreceptor tyrosine kinase-like orphan receptor, which functions in Wntligand signaling³⁰. Likewise, CD3E is a single-pass cell surfaceglycoprotein involved in antigen recognition³¹. CD9 is a 4-passtransmembrane protein from the tetraspanin family and it functions inintegrin signaling³². Finally, G-protein coupled receptors GPR40 andFrizzled-4 are 7-pass transmembrane receptors for long-chain free fattyacids and Wnt proteins, respectively³³′³⁴. Treatment of HEK 293T celllines stably expressing these transmembrane HaloTag fusion proteins withHyT13 efficiently induced their degradation (FIG. 12d ), demonstratingthe potential of our hydrophobic tagging system to degrade transmembraneproteins. These experiments show that fusions to either the amino orcarboxy terminus of the HaloTag protein are susceptible to this smallmolecule-induced degradation strategy and that transmembrane proteinscan be degraded by HyT13.

We also explored the possibility of employing the hydrophobic taggingsystem in the zebrafish Danio rerio. We injected HA-HaloTag-Smad5 cRNAinto zebrafish embryos and then treated the embryos with either vehicleor HyT13. Immunoblotting of injected embryo lysates revealed that thefusion protein is very efficiently degraded, demonstrating that HyT13 isable to penetrate the chorion and can direct the HaloTag fusion proteinsfor degradation in zebrafish (FIG. 12e ). These experiments show thatHyT13 is capable of degrading fusion proteins in various cell lines, aswell as in zebrafish embryos.

HyT13 Suppresses HaloTag-RasG12V Tumor Burden in Mice

We next explored the functional utility of HaloTag-based degradation ofan oncogene by HyT13 both in cell culture and in mice. The small GTPaseH-Ras is one of the most commonly mutated genes in cancer, with up to90% of cancers harboring activating mutations in this gene³⁵. Activatingmutations, such as the H-RasG12V allele, lead to decreased dependence onextracellular mitogenic signals. Ectopic expression of H-RasG12V inmouse fibroblast cell line NIH-3T3 can lead to a transformed phenotype,as demonstrated by assays in cell culture and in mice. When H-RasG12Vexpressing cells are grown in culture under low serum conditions theylose cell-to-cell contact inhibition and form distinct foci instead ofgrowing as a cellular monolayer. Furthermore, these transformed cellsare capable of tumor formation when injected into immuno-compromisednude mice³⁶′³⁷. We investigated whether (1) HaloTag-H-RasG12V drivenfocus formation can be suppressed in NIH-3T3 cells and (2)HaloTag-H-RasG12V driven tumor burden in mice can be reduced byadministration of HyT13. First, NIH-3T3 cells were stably infected witha HA-HaloTag-H-RasG12V retroviral construct. The encoded fusion proteinwas readily degraded with HyT13 (FIG. 13a ). To test the HaloTagreceptor specificity for HyT13, we generated a point mutation in theHaloTag protein (HaloTagD106A) that is unable to form a covalent bondwith the reactive chloroalkane in HyT13²⁶. Unlike HA-HaloTag-H-RasG12V,HA-HaloTag(D106A)-H-RasG12V fusion protein was unaffected by HyT13 (FIG.13a ). Next, we plated both cell lines sparsely (10⁵ cells/10-cm plate)in 10% FBS containing media. The next day, the media was replaced with1% FBS containing media and the cultures were treated with eithervehicle or HyT13. By day 6, both vehicle-treated cell lines andHyT13-treated HA-HaloTag(D106A)-H-RasG12V expressing cells had formedmany foci, whereas HA-HaloTag-H-RasG12V expressing cells treated withHyT13 had grown a normal monolayer of cells, much like the parentalNIH-3T3 cells (FIG. 13b-c ). In the absence of HyT13,HA-HaloTag-H-RasG12V expressing cells exhibited slightly higher numberof colonies than HA-HaloTag(D106A)-H-RasG12V cells. However, weattribute this observation to slight differences in retroviral infectionefficiencies, since we have observed instances where theHaloTag(D106A)-H-RasG12V cells exhibit more colonies than theHA-HaloTag-H-RasG12V cells as well (data not shown). These resultsdemonstrate that hydrophobic tagging can be used to reduce proteinactivity in the context of in vitro cell culture.

To examine whether the HaloTag:HyT13 based system could be used in mousemodels to relieve the H-RasG12V-driven tumor burden, we first evaluatedthe pharmacokinetics of HyT13. We performed a maximum tolerated doseexperiment with HyT13 in nude mice at doses up to 100 mg/kg over a14-day treatment regimen. No obvious phenotype was observed even at thehighest dose (FIG. 9). Next, we sought to determine the serumbioavailability of HyT13 following injections. HyT13 was administered at25 mg/kg by intraperitoneal (IP) injection into Swiss Webster mice andthe serum was collected at 1 and 24 hours post-injection. At 1 hour postHyT13 administration the blood serum concentration was approximately 2μM, and by 24 hours the HyT13 concentration had dropped to about 500 nM(FIG. 10). Based on our previous experiments in a cell culture setting,we speculated that these serum HyT13 concentrations would be sufficientto suppress H-RasG12V tumor formation in mice. To test this, we injectedNIH-3T3 cells expressing HA-HaloTag-H-RasG12V into the flank of nudemice and on the same day started a daily treatment regimen of vehicle,25 mg/kg HyT13 or 100 mg/kg HyT13. Obvious solid tumor masses wereobserved on day 9 in vehicle-treated mice and the tumor volume grewexponentially until day 13, when the animals were sacrificed. The tumorsin HyT13 mice were on average 6 times smaller than in vehicle treatedmice, suggesting that HyT13 was able to reduce H-RasG12V tumor formation(FIG. 13d ). These data clearly demonstrate the utility of theHaloTag:HyT13 system in perturbing protein function in live animals.

DISCUSSION

The present invention relates to a novel hydrophobic tagging technologyto systematically degrade levels of a specific protein upon addition ofa small molecule (FIG. 9). This strategy has several benefits over theexisting technologies. First, protein degradation is achieved uponcompound administration as opposed to following ligand withdrawal. Thisaspect is particularly relevant when a protein needs to be expressed forlong periods before the study, as there is no continuous ligandtreatment necessary to maintain expression of the POI. In contrast,DD-based methods (see Introduction) of controlling protein abundancerequire constant drug administration, which can be both time-consumingand expensive. Also, there are likely fluctuations in the concentrationof the fusion protein between ligand administrations using the DD-basedsystem, whereas the expression of the HaloTag fusion protein is stablein the absence of the degradation signal. Therefore, depending on theapplication, it can be desirable to have a system where the smallmolecule induces degradation, rather than stabilization, of the POI.Second, our HaloTag:HyT13 method relies on the single introduction of afusion domain to the POI. This feature contrasts with the auxin system,where an exogenous plant E3 ligase must be expressed in addition to thefusion protein. Third, almost all human and mouse genes are commerciallyavailable as both N- and C-terminal HaloTag fusions in transient andlentiviral expression vectors. These protein fusions with the 34 kDaHaloTag receptor are proving useful in many studies of protein functionsince they can be readily labeled in vivo and purified using fluorescentor biotinylated HaloTag reagents. The ability to degrade these fusionproteins with the hydrophobic tag HyT13 only adds to the repertoire ofpossible HaloTag applications. Although HyT13 is not yet commerciallyavailable, this small molecule can be obtained using standard syntheticmethods in four steps from commercially available starting materialswith an overall yield of 63% (Scheme 2, above).

One of the criticisms that surround the several FKBP12 based degradationsystems is their reliance on either rapamycin, FK506 or theirderivatives to cause protein perturbation. Since these are bioactivesmall molecules, they could induce biological effects unrelated toperturbing the POI. In contrast, HaloTag dehalogenase is a bacterialgene and covalent binding of HyT13 to HaloTag affords this system a highdegree of specificity. This bioorthogonality may explain the lack ofnoticeable HyT13 cytotoxicity even upon 1,000-fold administration overits IC₅₀ value of 21 nM in cell culture. Moreover, mice injected dailywith HyT13 at 100 mg/kg for 14 days gained weight normally, suggestingthat HyT13 possesses no in vivo toxicity even at this high dose.

Like several other systematic degradation methods, the HaloTag:HyT13methodology is not able to degrade endogenous proteins unless theHaloTag gene is fused with the gene of interest. However, there are twoviable strategies to overcome this limitation and subject endogenousproteins to Halotag:HyT13-mediated regulation in culture or liveanimals. First, it is possible to generate HaloTag fusion constructs viatargeted genome engineering. Recent advances in zinc fingernucleases^(20,38,39) and homologous recombination⁴⁰ technologies openthe possibility of systematically tagging endogenous proteins in rodentsin a manner similar to yeast. The second approach would be to inactivatethe endogenous gene by knockdown or knockout techniques and introducethe corresponding HaloTag fusion gene into the animal. Both approachesshould be amenable to bypassing an early requirement of an essentialgene, thus allowing the study of its function later during organogenesisor disease development.

In summary, herein we describe a chemical biology approach tosystematically degrade any POI in either cell culture or whole animals.The system requires construction of a single fusion protein, which isspecifically degraded by the addition of a non-toxic, low-molecularweight hydrophobic tag. We believe this system is particularly amenableto animal studies, as we have shown here with experiments in zebrafishand mice. Additionally, our findings suggest that hydrophobic taggingrepresents a novel approach to promote targeted degradation ofendogenous proteins independent of the HaloTag:HyT13 system.

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1. A compound of formula HYD-L_(R), wherein: HYD is a hydrophobic groupselected from the group consisting of HTL-5, HTL-14 and HTL-18:

and L_(R) is a linker group having a reactive moiety capable of forminga covalent link between the HYD and a target protein of interest.
 2. Thecompound of claim 1, wherein the reactive moiety is a haloalkyl groupoptionally substituted with a monoether or diether group.
 3. Thecompound of claim 1, wherein the reactive moiety is selected from thegroup consisting of:


4. The compound of claim 1, wherein the target protein of interestcomprises at least one selected from the group consisting of ahaloalkane dehalogenase, O6-alkylguanine-DNA alkyltransferase, ACPsynthase, SCP synthase, and SFP synthase.
 5. The compound of claim 1,wherein the HYD has a ClogP of at least about 4.0.
 6. The compound ofclaim 1, wherein the target protein of interest is at least one selectedfrom the group consisting of a structural protein, a receptor, anenzyme, a cell surface protein, a behavioral protein, a cell adhesionprotein and a protein involved in a biological activity, wherein thebiological activity is at least one selected from the group consistingof catalytic activity, aromatase activity, motor activity, helicaseactivity, metabolic processes, antioxidant activity, proteolysis,biosynthesis, kinase activity, oxidoreductase activity, transferaseactivity, hydrolase activity, lyase activity, isomerase activity, ligaseactivity, enzyme regulator activity, signal transducer activity,structural molecule activity, binding activity, cell motility, membranefusion, cell communication, regulation of biological processes,development, cell differentiation, response to stimulus, cell death,protein transporter activity, nuclear transport, ion transporteractivity, channel transporter activity, carrier activity, permeaseactivity, secretion activity, electron transporter activity,pathogenesis, chaperone regulator activity, nucleic acid bindingactivity, transcription regulator activity, extracellular organizationand biogenesis activity and translation regulator activity.
 7. A methodof determining whether a protein of interest is a target of a bioactiveagent or drug, the method comprising: exposing a cell that utilizes theprotein of interest to a protein of interest which is covalentlymodified with a compound of claim 1 and is present within or on thesurface of the cell, whereby the HYD induces degradation of the proteinof interest intracellularly or on the surface of cell; determining ifthe degradation of the covalently labeled protein modulates thebiological activity of the cell through a change in a phenotypicresponse of the cell, wherein, if the degradation of the covalentlylabeled protein modulates the biological activity of the cell through achange in a phenotypic response of the cell, the protein of interest isidentified as a target for a bioactive agent or drug that treats orprevents a disease and/or condition modulated through the protein ofinterest.
 8. A method of inducing degradation of a protein of interestin a cell, the method comprising reacting, intracellularly or on thesurface of the cell, the expressed protein of interest with a compoundcomprising the compound of claim 1, wherein the compound of claim 1forms a covalent bond with the protein of interest, thus yielding ahydrophobically labeled protein, which undergoes degradation.